Methods and apparatus for measuring analytes using large scale FET arrays

ABSTRACT

Methods and apparatus relating to very large scale FET arrays for analyte measurements. ChemFET (e.g., ISFET) arrays may be fabricated using conventional CMOS processing techniques based on improved FET pixel and array designs that increase measurement sensitivity and accuracy, and at the same time facilitate significantly small pixel sizes and dense arrays. Improved array control techniques provide for rapid data acquisition from large and dense arrays. Such arrays may be employed to detect a presence and/or concentration changes of various analyte types in a wide variety of chemical and/or biological processes. In one example, chemFET arrays facilitate DNA sequencing techniques based on monitoring changes in the concentration of inorganic pyrophosphate (PPi), hydrogen ions, and nucleotide triphosphates.

RELATED APPLICATIONS

This application is a national stage filing under 35 U.S.C. §371 of PCTInternational application PCT/US2009/003766, filed Jun. 25, 2009, PCTArticle 21(2) in English, which claims priority to patent applicationsGB 0811657.6 and GB 0811656.8, both filed on Jun. 25, 2008, the entirecontents of all of which are incorporated by reference.

FIELD OF THE DISCLOSURE

The present disclosure is directed generally to inventive methods andapparatus relating to detection and measurement of one or more analytesvia electronic sensors.

BACKGROUND

Electronic devices and components have found numerous applications inchemistry and biology (more generally, “life sciences”), especially fordetection and measurement of various chemical and biological reactionsand identification, detection and measurement of various compounds. Onesuch electronic device is referred to as an ion-sensitive field effecttransistor, often denoted in the relevant literature as ISFET (orpHFET). ISFETs conventionally have been explored, primarily in theacademic and research community, to facilitate measurement of thehydrogen ion concentration of a solution (commonly denoted as “pH”).

More specifically, an ISFET is an impedance transformation device thatoperates in a manner similar to that of a MOSFET (Metal OxideSemiconductor Field Effect Transistor), and is particularly configuredto selectively measure ion activity in a solution (e.g., hydrogen ionsin the solution are the “analytes”). A detailed theory of operation ofan ISFET is given in “Thirty years of ISFETOLOGY: what happened in thepast 30 years and what may happen in the next 30 years,” P. Bergveld,Sens. Actuators, 88 (2003), pp. 1-20, which publication is herebyincorporated herein by reference (hereinafter referred to as“Bergveld”).

FIG. 1 illustrates a cross-section of a p-type (p-channel) ISFET 50fabricated using a conventional CMOS (Complimentary Metal OxideSemiconductor) process. However, biCMOS (i.e., bipolar and CMOS)processing may also be used, such as a process that would include a PMOSFET array with bipolar structures on the periphery. Taking the CMOSexample, P-type ISFET fabrication is based on a p-type silicon substrate52, in which an n-type well 54 forming a transistor “body” is formed.Highly doped p-type (p+) regions S and D, constituting a source 56 and adrain 58 of the ISFET, are formed within the n-type well 54. A highlydoped n-type (n+) region B is also formed within the n-type well toprovide a conductive body (or “bulk”) connection 62 to the n-type well.An oxide layer 65 is disposed above the source, drain and bodyconnection regions, through which openings are made to provideelectrical connections (via electrical conductors) to these regions; forexample, metal contact 66 serves as a conductor to provide an electricalconnection to the drain 58, and metal contact 68 serves as a conductorto provide a common connection to the source 56 and n-type well 54, viathe highly conductive body connection 62. A polysilicon gate 64 isformed above the oxide layer at a location above a region 60 of then-type well 54, between the source 56 and the drain 58. Because it isdisposed between the polysilicon gate 64 and the transistor body (i.e.,the n-type well), the oxide layer 65 often is referred to as the “gateoxide.”

Like a MOSFET, the operation of an ISFET is based on the modulation ofcharge concentration caused by a MOS (Metal-Oxide-Semiconductor)capacitance constituted by the polysilicon gate 64, the gate oxide 65and the region 60 of the n-type well 54 between the source and thedrain. When a negative voltage is applied across the gate and sourceregions (V_(GS)<0 Volts), a “p-channel” 63 is created at the interfaceof the region 60 and the gate oxide 65 by depleting this area ofelectrons. This p-channel 63 extends between the source and the drain,and electric current is conducted through the p-channel when thegate-source potential V_(GS) is negative enough to attract holes fromthe source into the channel. The gate-source potential at which thechannel 63 begins to conduct current is referred to as the transistor'sthreshold voltage V_(TH) (the transistor conducts when V_(GS) has anabsolute value greater than the threshold voltage V_(TH)). The source isso named because it is the source of the charge carriers (holes for ap-channel) that flow through the channel 63; similarly, the drain iswhere the charge carriers leave the channel 63.

In the ISFET 50 of FIG. 1, the n-type well 54 (transistor body), via thebody connection 62, is forced to be biased at a same potential as thesource 56 (i.e., V_(SB)=0 Volts), as seen by the metal contact 68connected to both the source 56 and the body connection 62. Thisconnection prevents forward biasing of the p+ source region and then-type well, and thereby facilitates confinement of charge carriers tothe area of the region 60 in which the channel 63 may be formed. Anypotential difference between the source 56 and the body/n-type well 54(a non-zero source-to-body voltage V_(SB)) affects the threshold voltageV_(TH) of the ISFET according to a nonlinear relationship, and iscommonly referred to as the “body effect,” which in many applications isundesirable.

As also shown in FIG. 1, the polysilicon gate 64 of the ISFET 50 iscoupled to multiple metal layers disposed within one or more additionaloxide layers 75 disposed above the gate oxide 65 to form a “floatinggate” structure 70. The floating gate structure is so named because itis electrically isolated from other conductors associated with theISFET; namely, it is sandwiched between the gate oxide 65 and apassivation layer 72. In the ISFET 50, the passivation layer 72constitutes an ion-sensitive membrane that gives rise to theion-sensitivity of the device; i.e., the presence of—analytes such asions in an “analyte solution” 74 (i.e., a solution containing analytes(including ions) of interest or being tested for the presence ofanalytes of interest) in contact with the passivation layer 72,particularly in a sensitive area 78 above the floating gate structure70, alters the electrical characteristics of the ISFET so as to modulatea current flowing through the p-channel 63 between the source 56 and thedrain 58. The passivation layer 72 may comprise any one of a variety ofdifferent materials to facilitate sensitivity to particular ions; forexample, passivation layers comprising silicon nitride or siliconoxynitride, as well as metal oxides such as silicon, aluminum ortantalum oxides, generally provide sensitivity to hydrogen ionconcentration (pH) in the analyte solution 74, whereas passivationlayers comprising polyvinyl chloride containing valinomycin providesensitivity to potassium ion concentration in the analyte solution 74.Materials suitable for passivation layers and sensitive to other ionssuch as sodium, silver, iron, bromine, iodine, calcium, and nitrate, forexample, are known.

With respect to ion sensitivity, an electric potential difference,commonly referred to as a “surface potential,” arises at thesolid/liquid interface of the passivation layer 72 and the analytesolution 74 as a function of the ion concentration in the sensitive area78 due to a chemical reaction (e.g., usually involving the dissociationof oxide surface groups by the ions in the analyte solution 74 inproximity to the sensitive area 78). This surface potential in turnaffects the threshold voltage V_(TH) of the ISFET; thus, it is thethreshold voltage V_(TH) of the ISFET that varies with changes in ionconcentration in the analyte solution 74 in proximity to the sensitivearea 78.

FIG. 2 illustrates an electric circuit representation of the p-channelISFET 50 shown in FIG. 1. With reference again to FIG. 1, a referenceelectrode 76 (a conventional Ag/AgCl electrode) in the analyte solution74 determines the electric potential of the bulk of the analyte solution74 itself and is analogous to the gate terminal of a conventionalMOSFET, as shown in FIG. 2. In a linear or non-saturated operatingregion of the ISFET, the drain current I_(D) is given as:

$\begin{matrix}{{I_{D} = {{\beta\left( {V_{GS} - V_{TH} - {\frac{1}{2}V_{DS}}} \right)}V_{DS}}},} & (1)\end{matrix}$where V_(DS) is the voltage between the drain and the source, and β is atransconductance parameter (in units of Amps/Volts²) given by:

$\begin{matrix}{{\beta = {\mu\;{C_{ox}\left( \frac{W}{L} \right)}}},} & (2)\end{matrix}$where μ represents the carrier mobility, C_(ox) is the gate oxidecapacitance per unit area, and the ratio W/L is the width to lengthratio of the channel 63. If the reference electrode 76 provides anelectrical reference or ground (V_(G)=0 Volts), and the drain currentI_(D) and the drain-to-source voltage V_(DS) are kept constant,variations of the source voltage V_(S) of the ISFET directly trackvariations of the threshold voltage V_(TH), according to Eq. (1); thismay be observed by rearranging Eq. (1) as:

$\begin{matrix}{V_{S} = {{- V_{TH}} - {\left( {\frac{I_{D}}{\beta\; V_{DS}} + \frac{V_{DS}}{2}} \right).}}} & (3)\end{matrix}$

Since the threshold voltage V_(TH) of the ISFET is sensitive to ionconcentration as discussed above, according to Eq. (3) the sourcevoltage V_(S) provides a signal that is directly related to the ionconcentration in the analyte solution 74 in proximity to the sensitivearea 78 of the ISFET. More specifically, the threshold voltage V_(TH) isgiven by:

$\begin{matrix}{{V_{TH} = {V_{FB} - \frac{Q_{B}}{C_{ox}} + {2\phi_{F}}}},} & (4)\end{matrix}$where V_(FB) is the flatband voltage, Q_(B) is the depletion charge inthe silicon and φ_(F) is the Fermi-potential. The flatband voltage inturn is related to material properties such as workfunctions and chargeaccumulation. In the case of an ISFET, with reference to FIGS. 1 and 2,the flatband voltage contains terms that reflect interfaces between 1)the reference electrode 76 (acting as the transistor gate G) and theanalyte solution 74; and 2) the analyte solution 74 and the passivationlayer 72 in the sensitive area 78 (which in turn mimics the interfacebetween the polysilicon gate 64 of the floating gate structure 70 andthe gate oxide 65). The flatband voltage V_(FB) is thus given by:

$\begin{matrix}{{V_{FB} = {E_{ref} - \Psi_{0} + \chi_{sol} - \frac{\Phi_{Si}}{q} - \frac{Q_{ss} + Q_{ox}}{C_{ox}}}},} & (5)\end{matrix}$where E_(ref) is the reference electrode potential relative to vacuum,Ψ₀ is the surface potential that results from chemical reactions at theanalyte solution/passivation layer interface (e.g., dissociation ofsurface groups in the passivation layer), and χ_(sol) is the surfacedipole potential of the analyte solution 74. The fourth term in Eq. (5)relates to the silicon workfunction (q is the electron charge), and thelast term relates to charge densities at the silicon surface and in thegate oxide. The only term in Eq. (5) sensitive to ion concentration inthe analyte solution 74 is Ψ₀, as the ion concentration in the analytesolution 74 controls the chemical reactions (dissociation of surfacegroups) at the analyte solution/passivation layer interface. Thus,substituting Eq. (5) into Eq. (4), it may be readily observed that it isthe surface potential Ψ₀ that renders the threshold voltage V_(TH)sensitive to ion concentration in the analyte solution 74.

Regarding the chemical reactions at the analyte solution/passivationlayer interface, the surface of a given material employed for thepassivation layer 72 may include chemical groups that may donate protonsto or accept protons from the analyte solution 74, leaving at any giventime negatively charged, positively charged, and neutral sites on thesurface of the passivation layer 72 at the interface with the analytesolution 74. A model for this proton donation/acceptance process at theanalyte solution/passivation layer interface is referred to in therelevant literature as the “Site-Dissociation Model” or the“Site-Binding Model,” and the concepts underlying such a process may beapplied generally to characterize surface activity of passivation layerscomprising various materials (e.g., metal oxides, metal nitrides, metaloxynitrides).

Using the example of a metal oxide for purposes of illustration, thesurface of any metal oxide contains hydroxyl groups that may donate aproton to or accept a proton from the analyte to leave negatively orpositively charged sites, respectively, on the surface. The equilibriumreactions at these sites may be described by:AOH

AO⁻+H_(s) ⁺  (6)AOH₂ ⁺

AOH+H_(s) ⁺  (7)where A denotes an exemplary metal, H_(s) ⁺ represents a proton in theanalyte solution 74, Eq. (6) describes proton donation by a surfacegroup, and Eq. (7) describes proton acceptance by a surface group. Itshould be appreciated that the reactions given in Eqs. (6) and (7) alsoare present and need to be considered in the analysis of a passivationlayer comprising metal nitrides, together with the equilibrium reaction:ANH₃ ⁺

ANH₂+H⁺,  (7b)wherein Eq. (7b) describes another proton acceptance equilibriumreaction. For purposes of the present discussion however, again only theproton donation and acceptance reactions given in Eqs. (6) and (7) areinitially considered to illustrate the relevant concepts.

Based on the respective forward and backward reaction rate constants foreach equilibrium reaction, intrinsic dissociation constants K_(a) (forthe reaction of Eq. (6)) and K_(b) (for the reaction of Eq. (7)) may becalculated that describe the equilibrium reactions. These intrinsicdissociation constants in turn may be used to determine a surface chargedensity σ₀ (in units of Coulombs/unit area) of the passivation layer 72according to:σ₀ =−qB,  (8)where the term B denotes the number of negatively charged surface groupsminus the number of positively charged surface groups per unit area,which in turn depends on the total number of proton donor/acceptor sitesper unit area N_(S) on the passivation layer surface, multiplied by afactor relating to the intrinsic dissociation constants K_(a) and K_(b)of the respective proton donation and acceptance equilibrium reactionsand the surface proton activity (or pH_(S)). The effect of a smallchange in surface proton activity (pH_(S)) on the surface charge densityis given by:

$\begin{matrix}{{\frac{\partial\sigma_{0}}{\partial{pH}_{S}} = {{{- q}\frac{\partial B}{\partial{pH}_{S}}} = {{- q}\;\beta_{int}}}},} & (9)\end{matrix}$where β_(int) is referred to as the “intrinsic buffering capacity” ofthe surface. It should be appreciated that since the values of N_(S),K_(a) and K_(b) are material dependent, the intrinsic buffering capacityβ_(int) of the surface similarly is material dependent.

The fact that ionic species in the analyte solution 74 have a finitesize and cannot approach the passivation layer surface any closer thanthe ionic radius results in a phenomenon referred to as a “double layercapacitance” proximate to the analyte solution/passivation layerinterface. In the Gouy-Chapman-Stern model for the double layercapacitance as described in Bergveld, the surface charge density σ₀ isbalanced by an equal but opposite charge density in the analyte solution74 at some position from the surface of the passivation layer 72. Thesetwo parallel opposite charges form a so-called “double layercapacitance” C_(dl) (per unit area), and the potential difference acrossthe capacitance C_(dl) is defined as the surface potential Ψ₀, accordingto:σ₀ =C _(dl)Ψ₀=−σ_(dl)  (10)where σ_(dl) is the charge density on the analyte solution side of thedouble layer capacitance. This charge density σ_(dl) in turn is afunction of the concentration of all ion species or other analytespecies (i.e., not just protons) in the bulk analyte solution 74; inparticular, the surface charge density can be balanced not only byhydrogen ions but other ion species (e.g., Na⁺, K⁺) in the bulk analytesolution.

In the regime of relatively lower ionic strengths (e.g., <1 mole/liter),the Debye theory may be used to describe the double layer capacitanceC_(dl) according to:

$\begin{matrix}{C_{dl} = \frac{k\; ɛ_{0}}{\lambda}} & (11)\end{matrix}$where k is the dielectric constant ∈/∈₀ (for relatively lower ionicstrengths, the dielectric constant of water may be used), and λ is theDebye screening length (i.e., the distance over which significant chargeseparation can occur). The Debye length λ is in turn inverselyproportional to the square root of the strength of the ionic species inthe analyte solution, and in water at room temperature is given by:

$\begin{matrix}{\lambda = {\frac{0.3\mspace{14mu}{nm}}{\sqrt{I}}.}} & (12)\end{matrix}$The ionic strength I of the bulk analyte is a function of theconcentration of all ionic species present, and is given by:

$\begin{matrix}{{I = {\frac{1}{2}{\sum\limits_{s}{z_{s}^{2}c_{s}}}}},} & (13)\end{matrix}$where z_(s) is the charge number of ionic species s and c_(s) is themolar concentration of ionic species s. Accordingly, from Eqs. (10)through (13), it may be observed that the surface potential is largerfor larger Debye screening lengths (i.e., smaller ionic strengths).

The relation between pH values present at the analytesolution/passivation layer interface and in the bulk solution isexpressed in the relevant literature by Boltzman statistics with thesurface potential Ψ₀ as a parameter:

$\begin{matrix}{\left( {{pH}_{s} - {pH}_{B}} \right) = {\frac{q\;\Psi_{0}}{kT}.}} & (14)\end{matrix}$From Eqs. (9), (10) and (14), the sensitivity of the surface potentialΨ₀ particularly to changes in the bulk pH of the analyte solution (i.e.,“pH sensitivity”) is given by:

$\begin{matrix}{{\frac{{\Delta\Psi}_{0}}{\Delta{pH}} = {{- 2.3}\frac{kT}{q}\alpha}},} & (15)\end{matrix}$where the parameter α is a dimensionless sensitivity factor that variesbetween zero and one and depends on the double layer capacitance C_(dl)and the intrinsic buffering capacity of the surface β_(int) as discussedabove in connection with Eq. (9). In general, passivation layermaterials with a high intrinsic buffering capacity β_(int) render thesurface potential Ψ₀ less sensitive to concentration in the analytesolution 74 of ionic species other than protons (e.g., α is maximized bya large β_(int)). From Eq. (15), at a temperature T of 298 degreesKelvin, it may be appreciated that a theoretical maximum pH sensitivityof 59.2 mV/pH may be achieved at α=1. From Eqs. (4) and (5), as notedabove, changes in the ISFET threshold voltage V_(TH) directly trackchanges in the surface potential Ψ₀; accordingly, the pH sensitivity ofan ISFET given by Eq. (15) also may be denoted and referred to herein asΔV_(TH) for convenience. In exemplary conventional ISFETs employing asilicon nitride or silicon oxynitride passivation layer 72 forpH-sensitivity, pH sensitivities ΔV_(TH) (i.e., a change in thresholdvoltage with change in pH of the analyte solution 74) over a range ofapproximately 30 mV/pH to 60 mV/pH have been observed experimentally.

Another noteworthy metric in connection with ISFET pH sensitivityrelates to the bulk pH of the analyte solution 74 at which there is nonet surface charge density Ψ₀ and, accordingly, a surface potential Ψ₀of zero volts. This pH is referred to as the “point of zero charge” anddenoted as pH_(pzc). With reference again to Eqs. (8) and (9), like theintrinsic buffering capacity β_(int), pH_(pzc) is a material dependentparameter. From the foregoing, it may be appreciated that the surfacepotential at any given bulk pH_(B) of the analyte solution 74 may becalculated according to:

$\begin{matrix}{{\Psi_{0}\left( {pH}_{B} \right)} = {\left( {{pH}_{B} - {pH}_{pzc}} \right){\frac{{\Delta\Psi}_{0}}{\Delta{pH}}.}}} & (16)\end{matrix}$Table 1 below lists various metal oxides and metal nitrides and theircorresponding points of zero charge (pH_(pzc)), pH sensitivities(ΔV_(TH)), and theoretical maximum surface potential at a pH of 9:

TABLE 1 Oxide/ Theoretical Ψ₀ Metal Nitride pH_(pzc) ΔV_(TH) (mV/pH)(mV) @ pH = 9 Al Al₂O₃ 9.2  54.5 (35° C.) −11 Zr ZrO₂ 5.1 50 150 Ti TiO₂5.5 57.4-62.3 201 (32° C., pH 3- 11) Ta Ta₂O₅ 2.9, 2.8 62.87 (35° C.)384 Si Si₃N₄ 4.6, 6-7 56.94 (25° C.) 251 Si SiO₂ 2.1 43 297 Mo MoO₃1.8-2.1 48-59 396 W WO₂ 0.3, 0.43, 0.5 50 435

Prior research efforts to fabricate ISFETs for pH measurements based onconventional CMOS processing techniques typically have aimed to achievehigh signal linearity over a pH range from 1-14. Using an exemplarythreshold sensitivity of approximately 50 mV/pH, and considering Eq. (3)above, this requires a linear operating range of approximately 700 mVfor the source voltage V_(S). As discussed above in connection with FIG.1, the threshold voltage V_(TH) of ISFETs (as well as MOSFETs) isaffected by any voltage V_(SB) between the source and the body (n-typewell 54). More specifically, the threshold voltage V_(TH) is a nonlinearfunction of a nonzero source-to-body voltage V_(SB). Accordingly, so asto avoid compromising linearity due to a difference between the sourceand body voltage potentials (i.e., to mitigate the “body effect”), asshown in FIG. 1 the source 56 and body connection 62 of the ISFET 50often are coupled to a common potential via the metal contact 68. Thisbody-source coupling also is shown in the electric circuitrepresentation of the ISFET 50 shown in FIG. 2.

While the foregoing discussion relates primarily to a steady stateanalysis of ISFET response based on the equilibrium reactions given inEqs. (6) and (7), the transient or dynamic response of a conventionalISFET to an essentially instantaneous change in ionic strength of theanalyte solution 74 (e.g., a stepwise change in proton or other ionicspecies concentration) has been explored in some research efforts. Oneexemplary treatment of ISFET transient or dynamic response is found in“ISFET responses on a stepwise change in electrolyte concentration atconstant pH,” J. C. van Kerkof, J. C. T. Eijkel and P. Bergveld, Sensorsand Actuators B, 18-19 (1994), pp. 56-59, which is incorporated hereinby reference.

For ISFET transient response, a stepwise change in the concentration ofone or more ionic species in the analyte solution in turn essentiallyinstantaneously changes the charge density σ_(dl) on the analytesolution side of the double layer capacitance C_(dl). Because theinstantaneous change in charge density σ_(dl) is faster than thereaction kinetics at the surface of the passivation layer 72, thesurface charge density σ₀ initially remains constant, and the change inion concentration effectively results in a sudden change in the doublelayer capacitance C_(dl). From Eq. (10), it may be appreciated that sucha sudden change in the capacitance C_(dl) at a constant surface chargedensity σ₀ results in a corresponding sudden change in the surfacepotential Ψ₀. FIG. 2A illustrates this phenomenon, in which anessentially instantaneous or stepwise increase in ion concentration inthe analyte solution, as shown in the top graph, results in acorresponding change in the surface potential Ψ₀, as shown in the bottomgraph of FIG. 2A. After some time, as the passivation layer surfacegroups react to the stimulus (i.e., as the surface charge densityadjusts), the system returns to some equilibrium point, as illustratedby the decay of the ISFET response “pulse” 79 shown in the bottom graphof FIG. 2A. The foregoing phenomenon is referred to in the relevantliterature (and hereafter in this disclosure) as an “ion-step” response.

As indicated in the bottom graph of FIG. 2A, an amplitude ΔΨ₀ of theion-step response 79 may be characterized by:

$\begin{matrix}{{{\Delta\Psi}_{0} = {{\Psi_{1} - \Psi_{2}} = {{\frac{\sigma_{0}}{C_{{dl},1}} - \frac{\sigma_{0}}{C_{{dl},2}}} = {\Psi_{1}\left( {1 - \frac{C_{{dl},1}}{C_{{dl},2}}} \right)}}}},} & (17)\end{matrix}$where Ψ₁ is an equilibrium surface potential at an initial ionconcentration in the analyte solution, C_(dl,1) is the double layercapacitance per unit area at the initial ion concentration, Ψ₂ is thesurface potential corresponding to the ion-step stimulus, and C_(dl,2)is the double layer capacitance per unit area based on the ion-stepstimulus. The time decay profile 81 associated with the response 79 isdetermined at least in part by the kinetics of the equilibrium reactionsat the analyte solution/passivation layer interface (e.g., as given byEqs. (6) and (7) for metal oxides, and also Eq. (7b) for metalnitrides). One instructive treatment in this regard is provided by“Modeling the short-time response of ISFET sensors,” P. Woias et al.,Sensors and Actuators B, 24-25 (1995) 211-217 (hereinafter referred toas “Woias”), which publication is incorporated herein by reference.

In the Woias publication, an exemplary ISFET having a silicon nitridepassivation layer is considered. A system of coupled non-lineardifferential equations based on the equilibrium reactions given by Eqs.(6), (7), and (7a) is formulated to describe the dynamic response of theISFET to a step (essentially instantaneous) change in pH; morespecifically, these equations describe the change in concentration overtime of the various surface species involved in the equilibriumreactions, based on the forward and backward rate constants for theinvolved proton acceptance and proton donation reactions and how changesin analyte pH affect one or more of the reaction rate constants.Exemplary solutions, some of which include multiple exponentialfunctions and associated time constants, are provided for theconcentration of each of the surface ion species as a function of time.In one example provided by Woias, it is assumed that the proton donationreaction given by Eq. (6) dominates the transient response of thesilicon nitride passivation layer surface for relatively small stepchanges in pH, thereby facilitating a mono-exponential approximation forthe time decay profile 81 of the response 79 according to:Ψ₀(t)=ΔΨ₀ e ^(−t/τ,)  (18)where the exponential function essentially represents the change insurface charge density as a function of time. In Eq. (16), the timeconstant τ is both a function of the bulk pH and material parameters ofthe passivation layer, according to:τ=τ₀×10^(pH/2)  (19)where τ₀ denotes a theoretical minimum response time that only dependson material parameters. For silicon nitride, Woias provides exemplaryvalues for τ₀ on the order of 60 microseconds to 200 microseconds. Forpurposes of providing an illustrative example, using τ₀=60 microsecondsand a bulk pH of 9, the time constant τ given by Eq. (19) is 1.9seconds. Exemplary values for other types of passivation materials maybe found in the relevant literature and/or determined empirically.

Previous efforts to fabricate two-dimensional arrays of ISFETs based onthe ISFET design of FIG. 1 have resulted in a maximum of 256 ISFETsensor elements (or “pixels”) in an array (i.e., a 16 pixel by 16 pixelarray). Exemplary research in ISFET array fabrication is reported in thepublications “A large transistor-based sensor array chip for directextracellular imaging,” M. J. Milgrew, M. O. Riehle, and D. R. S.Cumming, Sensors and Actuators, B: Chemical, 111-112, (2005), pp.347-353, and “The development of scalable sensor arrays using standardCMOS technology,” M. J. Milgrew, P. A. Hammond, and D. R. S. Cumming,Sensors and Actuators, B: Chemical, 103, (2004), pp. 37-42, whichpublications are incorporated herein by reference and collectivelyreferred to hereafter as “Milgrew et al.” Other research effortsrelating to the realization of ISFET arrays are reported in thepublications “A very large integrated pH-ISFET sensor array chipcompatible with standard CMOS processes,” T. C. W. Yeow, M. R. Haskard,D. E. Mulcahy, H. I. Seo and D. H. Kwon, Sensors and Actuators B:Chemical, 44, (1997), pp. 434-440 and “Fabrication of a two-dimensionalpH image sensor using a charge transfer technique,” Hizawa, T., Sawada,K., Takao, H., Ishida, M., Sensors and Actuators, B: Chemical 117 (2),2006, pp. 509-515, which publications also are incorporated herein byreference.

FIG. 3 illustrates one column 85 _(j) of a two-dimensional ISFET arrayaccording to the design of Milgrew et al. The column 85 _(j) includessixteen (16) pixels 80 ₁ through 80 ₁₆ and, as discussed further belowin connection with FIG. 7, a complete two-dimensional array includessixteen (16) such columns 85 _(j) (j=1, 2, 3, . . . 16) arranged side byside. As shown in FIG. 3, a given column 85 _(j) includes a currentsource I_(SOURCEj) that is shared by all pixels of the column, and ISFETbias/readout circuitry 82 _(j) (including current sink I_(SINKj)) thatis also shared by all pixels of the column. Each ISFET pixel 80 ₁through 80 ₁₆ includes a p-channel ISFET 50 having an electricallycoupled source and body (as shown in FIGS. 1 and 2), plus two switchesS1 and S2 that are responsive to one of sixteen row select signals(RSEL₁ through RSEL₁₆, and their complements). As discussed below inconnection with FIG. 7, a row select signal and its complement aregenerated simultaneously to “enable” or select a given pixel of thecolumn 85 _(j), and such signal pairs are generated in some sequence tosuccessively enable different pixels of the column one at a time.

As shown in FIG. 3, the switch S2 of each pixel 80 in the design ofMilgrew et al. is implemented as a conventional n-channel MOSFET thatcouples the current source I_(SOURCEj) to the source of the ISFET 50upon receipt of the corresponding row select signal. The switch S1 ofeach pixel 80 is implemented as a transmission gate, i.e., a CMOS pairincluding an n-channel MOSFET and a p-channel MOSFET, that couples thesource of the ISFET 50 to the bias/readout circuitry 82 _(j) uponreceipt of the corresponding row select signal and its complement. Anexample of the switch S1 ₁ of the pixel 80 ₁ is shown in FIG. 4, inwhich the p-channel MOSFET of the transmission gate is indicated as S1_(1P) and the n-channel MOSFET is indicated as S1 _(1N). In the designof Milgrew et al., a transmission gate is employed for the switch S1 ofeach pixel so that, for an enabled pixel, any ISFET source voltagewithin the power supply range V_(DD) to V_(SS) may be applied to thebias/readout circuitry 82 _(j) and output by the column as the signalV_(Sj). From the foregoing, it should be appreciated that each pixel 80in the ISFET sensor array design of Milgrew et al. includes fourtransistors, i.e., a p-channel ISFET, a CMOS-pair transmission gateincluding an n-channel MOSFET and a p-channel MOSFET for switch S1, andan n-channel MOSFET for switch S2.

As also shown in FIG. 3, the bias/readout circuitry 82 _(j) employs asource-drain follower configuration in the form of a Kelvin bridge tomaintain a constant drain-source voltage V_(DSj) and isolate themeasurement of the source voltage V_(Sj) from the constant drain currentI_(SOURCEj) for the ISFET of an enabled pixel in the column 85 _(j). Tothis end, the bias/readout circuitry 82 _(j) includes two operationalamplifiers A1 and A2, a current sink I_(SINKj), and a resistor R_(SDj).The voltage developed across the resistor R_(SDj) due to the currentI_(SINKj) flowing through the resistor is forced by the operationalamplifiers to appear across the drain and source of the ISFET of anenabled pixel as a constant drain-source voltage V_(DSj). Thus, withreference again to Eq. (3), due to the constant V_(DSj) and the constantI_(SOURCEj), the source voltage V_(Sj) of the ISFET of the enabled pixelprovides a signal corresponding to the ISFETs threshold voltage V_(TH),and hence a measurement of pH in proximity to the ISFETs sensitive area(see FIG. 1). The wide dynamic range for the source voltage V_(Sj)provided by the transmission gate S1 ensures that a full range of pHvalues from 1-14 may be measured, and the source-body connection of eachISFET ensures sufficient linearity of the ISFETs threshold voltage overthe full pH measurement range.

In the column design of Milgrew et al. shown in FIG. 3, it should beappreciated that for the Kelvin bridge configuration of the columnbias/readout circuitry 82 _(j) to function properly, a p-channel ISFET50 as shown in FIG. 1 must be employed in each pixel; more specifically,an alternative implementation based on the Kelvin bridge configurationis not possible using an n-channel ISFET. With reference again to FIG.1, for an n-channel ISFET based on a conventional CMOS process, then-type well 54 would not be required, and highly doped n-type regionsfor the drain and source would be formed directly in the p-type siliconsubstrate 52 (which would constitute the transistor body). For n-channelFET devices, the transistor body typically is coupled to electricalground. Given the requirement that the source and body of an ISFET inthe design of Milgrew et al. are electrically coupled together tomitigate nonlinear performance due to the body effect, this would resultin the source of an n-channel ISFET also being connected to electricalground (i.e., V_(S)=V_(B)=0 Volts), thereby precluding any useful outputsignal from an enabled pixel. Accordingly, the column design of Milgrewet al. shown in FIG. 3 requires p-channel ISFETs for proper operation.

It should also be appreciated that in the column design of Milgrew etal. shown in FIG. 3, the two n-channel MOSFETs required to implement theswitches S1 and S2 in each pixel cannot be formed in the n-type well 54shown in FIG. 1, in which the p-channel ISFET for the pixel is formed;rather, the n-channel MOSFETs are formed directly in the p-type siliconsubstrate 52, beyond the confines of the n-type well 54 for the ISFET.FIG. 5 is a diagram similar to FIG. 1, illustrating a widercross-section of a portion of the p-type silicon substrate 52corresponding to one pixel 80 of the column 85 j shown in FIG. 3, inwhich the n-type well 54 containing the drain 58, source 56 and bodyconnection 62 of the ISFET 50 is shown alongside a first n-channelMOSFET corresponding to the switch S2 and a second n-channel MOSFET S1_(1N) constituting one of the two transistors of the transmission gateS1 ₁ shown in FIG. 4.

Furthermore, in the design of Milgrew et al., the p-channel MOSFETrequired to implement the transmission gate S1 in each pixel (e.g., seeS1 _(1P) in FIG. 4) cannot be formed in the same n-type well in whichthe p-channel ISFET 50 for the pixel is formed. In particular, becausethe body and source of the p-channel ISFET are electrically coupledtogether, implementing the p-channel MOSFET S1 _(1P) in the same n-wellas the p-channel ISFET 50 would lead to unpredictable operation of thetransmission gate, or preclude operation entirely. Accordingly, twoseparate n-type wells are required to implement each pixel in the designof Milgrew et al. FIG. 6 is a diagram similar to FIG. 5, showing across-section of another portion of the p-type silicon substrate 52corresponding to one pixel 80, in which the n-type well 54 correspondingto the ISFET 50 is shown alongside a second n-type well 55 in which isformed the p-channel MOSFET S1 _(1P) constituting one of the twotransistors of the transmission gate S1 ₁ shown in FIG. 4. It should beappreciated that the drawings in FIGS. 5 and 6 are not to scale and maynot exactly represent the actual layout of a particular pixel in thedesign of Milgrew et al.; rather these figures are conceptual in natureand are provided primarily to illustrate the requirements of multiplen-wells, and separate n-channel MOSFETs fabricated outside of then-wells, in the design of Milgrew et al.

The array design of Milgrew et al. was implemented using a 0.35micrometer (μm) conventional CMOS fabrication process. In this process,various design rules dictate minimum separation distances betweenfeatures. For example, according to the 0.35 μm CMOS design rules, withreference to FIG. 6, a distance “a” between neighboring n-wells must beat least three (3) micrometers. A distance “a/2” also is indicated inFIG. 6 to the left of the n-well 54 and to the right of the n-well 55 toindicate the minimum distance required to separate the pixel 80 shown inFIG. 6 from neighboring pixels in other columns to the left and right,respectively. Additionally, according to typical 0.35 μm CMOS designrules, a distance “b” shown in FIG. 6 representing the width incross-section of the n-type well 54 and a distance “c” representing thewidth in cross-section of the n-type well 55 are each on the order ofapproximately 3 μm to 4 μm (within the n-type well, an allowance of 1.2μm is made between the edge of the n-well and each of the source anddrain, and the source and drain themselves have a width on the order of0.7 μm). Accordingly, a total distance “d” shown in FIG. 6 representingthe width of the pixel 80 in cross-section is on the order ofapproximately 12 μm to 14 μm. In one implementation, Milgrew et al.report an array based on the column/pixel design shown in FIG. 3comprising geometrically square pixels each having a dimension of 12.8μm by 12.8 μm.

In sum, the ISFET pixel design of Milgrew et al. is aimed at ensuringaccurate hydrogen ion concentration measurements over a pH range of1-14. To ensure measurement linearity, the source and body of eachpixel's ISFET are electrically coupled together. To ensure a full rangeof pH measurements, a transmission gate S1 is employed in each pixel totransmit the source voltage of an enabled pixel. Thus, each pixel ofMilgrew's array requires four transistors (p-channel ISFET, p-channelMOSFET, and two n-channel MOSFETs) and two separate. n-wells (FIG. 6).Based on a 0.35 micrometer conventional CMOS fabrication process andcorresponding design rules, the pixels of such an array have a minimumsize appreciably greater than 10 μm, i.e., on the order of approximately12 μm to 14 μm.

FIG. 7 illustrates a complete two-dimensional pixel array 95 accordingto the design of Milgrew et al., together with accompanying row andcolumn decoder circuitry and measurement readout circuitry. The array 95includes sixteen columns 85 ₁ through 85 ₁₆ of pixels, each columnhaving sixteen pixels as discussed above in connection with FIG. 3(i.e., a 16 pixel by 16 pixel array). A row decoder 92 provides sixteenpairs of complementary row select signals, wherein each pair of rowselect signals simultaneously enables one pixel in each column 85 ₁through 85 ₁₆ to provide a set of column output signals from the array95 based on the respective source voltages V_(S1) through V_(S16) of theenabled row of ISFETs. The row decoder 92 is implemented as aconventional four-to-sixteen decoder (i.e., a four-bit binary inputROW₁-ROW₄ to select one of 2⁴ outputs). The set of column output signalsV_(S1) through V_(S16) for an enabled row of the array is applied toswitching logic 96, which includes sixteen transmission gates S1 throughS16 (one transmission gate for each output signal). As above, eachtransmission gate of the switching logic 96 is implemented using ap-channel MOSFET and an n-channel MOSFET to ensure a sufficient dynamicrange for each of the output signals V_(S1) through V_(S16). The columndecoder 94, like the row decoder 92, is implemented as a conventionalfour-to-sixteen decoder and is controlled via the four-bit binary inputCOL₁-COL₄ to enable one of the transmission gates S1 through S16 of theswitching logic 96 at any given time, so as to provide a single outputsignal V_(S) from the switching logic 96. This output signal V_(S) isapplied to a 10-bit analog to digital converter (ADC) 98 to provide adigital representation D₁-D₁₀ of the output signal V_(S) correspondingto a given pixel of the array.

As noted earlier, individual ISFETs and arrays of ISFETs similar tothose discussed above have been employed as sensing devices in a varietyof chemical and biological applications. In particular, ISFETs have beenemployed as pH sensors in the monitoring of various processes involvingnucleic acids such as DNA. Some examples of employing ISFETs in variouslife-science related applications are given in the followingpublications, each of which is incorporated herein by reference: MassimoBarbaro, Annalisa Bonfiglio, Luigi Raffo, Andrea Alessandrini, PaoloFacci and Imrich Barák, “Fully electronic DNA hybridization detection bya standard CMOS biochip,” Sensors and Actuators B: Chemical, Volume 118,Issues 1-2, 2006, pp. 41-46; Toshinari Sakurai and Yuzuru Husimi,“Real-time monitoring of DNA polymerase reactions by a micro ISFET pHsensor,” Anal. Chem., 64(17), 1992, pp 1996-1997; S. Purushothaman, C.Toumazou, J. Georgiou, “Towards fast solid state DNA sequencing,”Circuits and Systems, vol. 4, 2002, pp. IV-169 to IV-172; S.Purushothaman, C. Toumazou, C. P. Ou, “Protons and single nucleotidepolymorphism detection: A simple use for the Ion Sensitive Field EffectTransistor,” Sensors and Actuators B: Chemical, Vol. 114, no. 2, 2006,pp. 964-968; A. L. Simonian, A. W. Flounders, J. R. Wild, “FET-BasedBiosensors for The Direct Detection of Organophosphate Neurotoxins,”Electroanalysis, Vol. 16, No. 22, 2004, pp. 1896-1906; C. Toumazou, S.Purushothaman, “Sensing Apparatus and Method,” United States PatentApplication 2004-0134798, published Jul. 15, 2004; and T. W. Koo, S.Chan, X. Su, Z. Jingwu, M. Yamakawa, V. M. Dubin, “Sensor Arrays andNucleic Acid Sequencing Applications,” United States Patent Application2006-0199193, published Sep. 7, 2006.

In general, the development of rapid and sensitive nucleic acidsequencing methods utilizing automated DNA sequencers has significantlyadvanced the understanding of biology. The term “sequencing” refers tothe determination of a primary structure (or primary sequence) of anunbranched biopolymer, which results in a symbolic linear depictionknown as a “sequence” that succinctly summarizes much of theatomic-level structure of the sequenced molecule. “DNA sequencing”particularly refers to the process of determining the nucleotide orderof a given DNA fragment. Analysis of entire genomes of viruses,bacteria, fungi, animals and plants is now possible, but such analysisgenerally is limited due to the cost and time required to sequence suchlarge genomes. Moreover, present conventional sequencing methods arelimited in terms of their accuracy, the length of individual templatesthat can be sequenced, and the rate of sequence determination.

Despite improvements in sample preparation and sequencing technologies,none of the present conventional sequencing strategies, including thoseto date that may involve ISFETs, has provided the cost reductionsrequired to increase throughput to levels required for analysis of largenumbers of individual human genomes. The ability to sequence many humangenomes facilitates an analysis of the genetic basis underlying disease(e.g., such as cancer) and aging, for example. Some recent efforts havemade significant gains in both the ability to prepare genomes forsequencing and to sequence large numbers of templates simultaneously.However, these and other efforts are still limited by the relativelylarge size of the reaction volumes needed to prepare templates that aredetectable by these systems, as well as the need for special nucleotideanalogues, and complex enzymatic or fluorescent methods to “read out”nucleotide sequence.

SUMMARY OF INVENTION

We have recognized and appreciated that large arrays of ISFETs may beparticularly configured and employed to facilitate DNA sequencingtechniques based on monitoring changes in chemical processes, includingDNA synthesis. More generally, Applicants have recognized andappreciated that large arrays of chemically-sensitive FETs (i.e.,chemFETs) may be employed to detect and measure static and/or dynamicconcentrations/levels of a variety of analytes (e.g., hydrogen ions,other ions, non-ionic molecules or compounds, etc.) in a host ofchemical and/or biological processes (e.g., biological or chemicalreactions, cell or tissue cultures or monitoring, neural activity,nucleic acid sequencing, etc.) in which valuable information may beobtained based on such analyte measurements.

Accordingly, various embodiments of the present disclosure are directedgenerally to inventive methods and apparatus relating to large scale FETarrays for measuring one or more analytes. In the various embodimentsdisclosed herein, FET arrays include multiple chemFETs, that act aschemical sensors. An ISFET, as discussed above, is a particular type ofchemFET that is configured for ion detection, and ISFETs may be employedin various embodiments disclosed herein. Other types of chemFETscontemplated by the present disclosure include enzyme FETs (EnFETs)which employ enzymes to detect analytes. It should be appreciated,however, that the present disclosure is not limited to ISFETs andEnFETs, but more generally relates to any FET that is configured forsome type of chemical sensitivity. As used herein, chemical sensitivitybroadly encompasses sensitivity to any molecule of interest, includingwithout limitation organic, inorganic, naturally occurring,non-naturally occurring, and synthetic chemical and biologicalcompounds, such as ions, small molecules, polymers such as nucleicacids, proteins, peptides, polysaccharides, and the like.

According to yet other embodiments, the present disclosure is directedgenerally to inventive methods and apparati relating to the use of theabove-described large scale chemFET arrays in the analysis of chemicalor biological samples. These samples are typically liquid (or aredissolved in a liquid) and of small volume, to facilitate high-speed,high-density determination of analyte (e.g., ion or other constituent)presence, concentration or other measurements on the analyte.

For example, some embodiments are directed to a “very large scale”two-dimensional chemFET sensor array (e.g., greater than 256 sensors),in which one or more chemFET-containing elements or “pixels”constituting the sensors of such an array are configured to monitor oneor more independent biological or chemical reactions or events occurringin proximity to the pixels of the array. In some exemplaryimplementations, the array may be coupled to one or more microfluidicsstructures that form one or more reaction chambers, or “wells” or“microwells,” over individual sensors or groups of sensors of the array,and apparatus which delivers analyte samples (i.e., analyte solutions)to the wells and removes them from the wells between measurements. Evenwhen microwells are not employed, the sensor array may be coupled to oneor more microfluidics structures for the delivery of one or moreanalytes to the pixels and for removal of analyte(s) betweenmeasurements. Accordingly, inventive aspects of this disclosure, whichare desired to be protected, include the various microfluidic structureswhich may be employed to flow analytes and where appropriate otheragents useful in for example the detection and measurement of analytesto and from the wells or pixels, the methods of manufacture of the arrayof wells, methods and structures for coupling the arrayed wells witharrayed pixels, and methods and apparatus for loading the wells withsample to be analyzed, including for example loading the wells withDNA-bearing beads when the apparatus is used for DNA sequencing orrelated analysis, as will be discussed in greater detail below.

In association with the microfluidics, unique reference electrodes andtheir coupling to the flow cell are also shown.

In various aspects of the invention, an analyte of particular interestis a byproduct of nucleic acid synthesis. Such a byproduct can bemonitored as the readout of a sequencing-by-synthesis method. Oneparticularly important byproduct is inorganic pyrophosphate (PPi) whichis released upon the addition (or incorporation) of a deoxynucleotidetriphosphate (also referred to herein as dNTP) to the 3′ end of anucleic acid (such as a sequencing primer). PPi may be hydrolyzed toorthophosphate (Pi) and free hydrogen ion (H⁺) in the presence of water(and optionally and far more rapidly in the presence ofpyrophosphatase). As a result, nucleotide incorporation, and thus asequencing-by-synthesis reaction, can be monitored by detecting PPi, Piand/or H⁺. Conventionally, PPi has not been detected or measured bychemFETs. Optically based sequencing-by-synthesis methods have detectedPPi via its sulfurylase-mediated conversion to adenosine triphosphate(ATP), and then luciferase-mediated conversion of luciferin tooxyluciferin in the presence of the previously generated ATP, withconcomitant release of light. Such detection is referred to herein as“enzymatic” detection of PPi. The invention provides methods fordetecting PPi using non-enzymatic methods. As used herein, non-enzymaticdetection of PPi is detection of PPi that does not require an enzymeother than any enzyme required to produce or release the PPi in thefirst instance (e.g., a polymerase). An example of non-enzymaticdetection of PPi is a detection method that does not require conversionof PPi to ATP.

H+ has been detected by measuring pH changes using standard pH meters orin some instances ISFETs. Importantly, many if not all prior attempts todetect nucleotide incorporation using ISFETs have focused solely on pHchanges and not on detection or measurement of released PPi.

The instant invention contemplates and thus provides methods formonitoring nucleic acid sequencing reactions and thus determining thenucleotide sequence of nucleic acids by detecting H⁺ (or changes in pH),PPi (or Pi) in the absence of presence of PPi (or Pi) specificreceptors, unincorporated dNTP, or some combination thereof. Someaspects therefore are aimed at monitoring pH while others are aimed atmonitoring (and detecting) ion pulses at the FET surface resulting fromchanges in ionic species in the solution in contact with such surface.

Thus, various aspects of the invention provide methods and apparati fordirectly detecting released PPi as an indicator of nucleotideincorporation into a nucleic acid. The invention does so through the useof the chemFET arrays described herein. In some embodiments, a nucleicacid synthesis reaction is performed in a solution that is in contactwith the chemFET and the released PPi is detected (or sensed) by thechemFET surface. Importantly, given the ability of the chemFETs of theinvention to detect PPi directly, such synthesis and/or sequencingreactions may be performed, and in some instances are preferablyperformed, in an environment that is substantially pH insensitive (i.e.,an environment in which changes in pH are not detected due to forexample the strong buffering capacity of the environment).

In other embodiments, the analyte of interest is hydrogen ion, and largescale ISFET arrays according to the present disclosure are specificallyconfigured to measure changes in H⁺ concentration (i.e., changes in pH).

In other embodiments, other biological or chemical reactions may bemonitored, and the chemFET arrays may be specifically configured tomeasure hydrogen ions and/or one or more other analytes that providerelevant information relating to the occurrence and/or progress of aparticular biological or chemical process of interest.

In various aspects, the chemFET arrays may be fabricated usingconventional CMOS (or biCMOS or other suitable) processing technologies,and are particularly configured to facilitate the rapid acquisition ofdata from the entire array (scanning all of the pixels to obtaincorresponding pixel output signals).

With respect to analyte detection and measurement, it should beappreciated that in various embodiments discussed in greater detailbelow, one or more analytes measured by a chemFET array according to thepresent disclosure may include any of a variety of biological orchemical substances that provide relevant information regarding abiological or chemical process (e.g., binding events such ashybridization of nucleic acids to each other, antigen-antibody binding,receptor-ligand binding, enzyme-inhibitor binding, enzyme-substratebinding, and the like). In some aspects, the ability to measure absoluteor relative as well as static and/or dynamic levels and/orconcentrations of one or more analytes, in addition to merelydetermining the presence or absence of an analyte, provides valuableinformation in connection with biological and chemical processes. Inother aspects, mere determination of the presence or absence of ananalyte or analytes of interest may provide valuable information may besufficient.

A chemFET array according to various inventive embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes. In one embodiment, one or more chemFETs of anarray may be particularly configured for sensitivity to one or moreanalytes, and in other embodiments different chemFETs of a given arraymay be configured for sensitivity to different analytes. For example, inone embodiment, one or more sensors (pixels) of the array may include afirst type of chemFET configured to be sensitive to a first analyte, andone or more other sensors of the array may include a second type ofchemFET configured to be sensitive to a second analyte different fromthe first analyte. In one embodiment, the first and second analytes maybe related to each other. As an example, the first and second analytesmay be byproducts of the same biological or chemical reaction/processand therefore they may be detected concurrently to confirm theoccurrence of a reaction (or lack thereof). Such redundancy ispreferably in some analyte detection methods. Of course, it should beappreciated that more than two different types of chemFETs may beemployed in any given array to detect and/or measure different types ofanalytes, and optionally to monitor biological or chemical processessuch as binding events. In general, it should be appreciated in any ofthe embodiments of sensor arrays discussed herein that a given sensorarray may be “homogeneous” and thereby consist of chemFETs ofsubstantially similar or identical type that detect and/or measure thesame analyte (e.g., pH or other ion concentration), or a sensor arraymay be “heterogeneous” and include chemFETs of different types to detectand/or measure different analytes. In another embodiment, the sensors inan array may be configured to detect and/or measure a single type (orclass) of analyte even though the species of that type (or class)detected and/or measured may be different between sensors. As anexample, all the sensors in an array may be configured to detect and/ormeasure nucleic acids, but each sensor detects and/or measures adifferent nucleic acid.

In yet other aspects, Applicants have specifically improved upon theISFET array design of Milgrew et al. discussed above in connection withFIGS. 1-7, as well as other conventional ISFET array designs, so as tosignificantly reduce pixel size, and thereby increase the number ofpixels of a chemFET array for a given semiconductor die size (i.e.,increase pixel density). In various embodiments, this increase in pixeldensity is accomplished while at the same time increasing thesignal-to-noise ratio (SNR) of output signals corresponding to monitoredbiological and chemical processes, and the speed with which such outputsignals may be read from the array. In particular, Applicants haverecognized and appreciated that by relaxing requirements for chemFETlinearity and focusing on a more limited measurement output signal range(e.g., output signals corresponding to a pH range of from approximately7 to 9 or smaller, rather than 1 to 14, as well as output signals thatdo not necessarily relate significantly to pH), individual pixelcomplexity and size may be significantly reduced, thereby facilitatingthe realization of very large scale dense chemFET arrays. Applicantshave also recognized and appreciated that alternative less complexapproaches to pixel selection in an chemFET array (e.g., alternatives tothe row and column decoder approach employed in the design of Milgrew etal. as shown in FIG. 7, whose complexity scales with array size), aswell as various data processing techniques involving ISFET responsemodeling and data extrapolation based on such modeling, facilitate rapidacquisition of data from significantly large and dense arrays.

With respect to chemFET array fabrication, Applicants have furtherrecognized and appreciated that various techniques employed in aconventional CMOS fabrication process, as well as variouspost-fabrication processing steps (wafer handling, cleaning, dicing,packaging, etc.), may in some instances adversely affect performance ofthe resulting chemFET array. For example, with reference again to FIG.1, one potential issue relates to trapped charge that may be induced inthe gate oxide 65 during etching of metals associated with the floatinggate structure 70, and how such trapped charge may affect chemFETthreshold voltage V_(TH). Another potential issue relates to thedensity/porosity of the chemFET passivation layer (e.g., see ISFETpassivation layer 72 in FIG. 1) resulting from low-temperature materialdeposition processes commonly employed in aluminum metal-based CMOSfabrication. While such low-temperature processes generally provide anadequate passivation layer for conventional CMOS devices, they mayresult in a somewhat low-density and porous passivation layer which maybe potentially problematic for chemFETs in contact with an analytesolution; in particular, a low-density porous passivation layer overtime may absorb and become saturated with analytes or other substancesin the solution, which may in turn cause an undesirable time-varyingdrift in the chemFETs threshold voltage V_(TH). This phenomenon may inturn impede accurate measurements of one or more particular analytes ofinterest. In view of the foregoing, other inventive embodimentsdisclosed herein relate to methods and apparatuses which mitigatepotentially adverse effects on chemFET performance that may arise fromvarious aspects of fabrication and post-fabrication processing/handlingof chemFET arrays.

Accordingly, one embodiment of the present invention is directed to anapparatus, comprising an array of CMOS-fabricated sensors, each sensorcomprising one chemically-sensitive field effect transistor (chemFET)and occupying an area on a surface of the array of 10 μm² or less.

Another embodiment is directed to a sensor array, comprising atwo-dimensional array of electronic sensors including at least 512 rowsand at least 512 columns of the electronic sensors, each sensorcomprising one chemically-sensitive field effect transistor (chemFET)configured to provide at least one output signal representing a presenceand/or concentration of an analyte proximate to a surface of thetwo-dimensional array.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising one chemically-sensitivefield effect transistor (chemFET). The array of CMOS-fabricated sensorsincludes more than 256 sensors, and a collection of chemFET outputsignals from all chemFETs of the array constitutes a frame of data. Theapparatus further comprises control circuitry coupled to the array andconfigured to generate at least one array output signal to providemultiple frames of data from the array at a frame rate of at least 1frame per second. In one aspect, the frame rate may be at least 10frames per second. In another aspect, the frame rate may be at least 20frames per second. In yet other aspects, the frame rate may be at least30, 40, 50, 70 or up to 100 frames per second.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The chemFET comprises a floating gatestructure, and a source and a drain having a first semiconductor typeand fabricated in a region having a second semiconductor type, whereinthere is no electrical conductor that electrically connects the regionhaving the second semiconductor type to either the source or the drain.

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor consisting of three field effecttransistors (FETs) including one chemically-sensitive field effecttransistor (chemFET).

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor comprising three or fewer field effecttransistors (FETs), wherein the three or fewer FETs includes onechemically-sensitive field effect transistor (chemFET).

Another embodiment is directed to an apparatus, comprising an array ofelectronic sensors, each sensor comprising a plurality of field effecttransistors (FETs) including one chemically-sensitive field effecttransistor (chemFET), and a plurality of electrical conductorselectrically connected to the plurality of FETs, wherein the pluralityof FETs are arranged such that the plurality of electrical conductorsincludes no more than four conductors traversing an area occupied byeach sensor and interconnecting multiple sensors of the array.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a plurality of fieldeffect transistors (FETs) including one chemically-sensitive fieldeffect transistor (chemFET), wherein all of the FETs in each sensor areof a same channel type and are implemented in a single semiconductorregion of an array substrate.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one and in someinstances at least two output signals representing a presence and/or aconcentration of an analyte proximate to a surface of the array. Foreach column of the plurality of columns, the array further comprisescolumn circuitry configured to provide a constant drain current and aconstant drain-to-source voltage to respective chemFETs in the column,the column circuitry including two operational amplifiers and adiode-connected FET arranged in a Kelvin bridge configuration with therespective chemFETs to provide the constant drain-to-source voltage.

Another embodiment is directed to a sensor array, comprising a pluralityof electronic sensors arranged in a plurality of rows and a plurality ofcolumns. Each sensor comprises one chemically-sensitive field effecttransistor (chemFET) configured to provide at least one output signaland in some instances at least two output signals representing aconcentration of ions in a solution proximate to a surface of the array.The array further comprises at least one row select shift register toenable respective rows of the plurality of rows, and at least one columnselect shift register to acquire chemFET output signals from respectivecolumns of the plurality of columns.

Another embodiment is directed to an apparatus, comprising an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The chemFET comprises a floating gatestructure, and a source and a drain having a first semiconductor typeand fabricated in a region having a second semiconductor type, whereinthere is no electrical conductor that electrically connects the regionhaving the second semiconductor type to either the source or the drain.The array includes a two-dimensional array of at least 512 rows and atleast 512 columns of the CMOS-fabricated sensors. Each sensor consistsof three field effect transistors (FETs) including the chemFET, and eachsensor includes a plurality of electrical conductors electricallyconnected to the three FETs. The three FETs are arranged such that theplurality of electrical conductors includes no more than four conductorstraversing an area occupied by each sensor and interconnecting multiplesensors of the array. All of the FETs in each sensor are of a samechannel type and implemented in a single semiconductor region of anarray substrate. A collection of chemFET output signals from allchemFETs of the array constitutes a frame of data. The apparatus furthercomprises control circuitry coupled to the array and configured togenerate at least one array output signal to provide multiple frames ofdata from the array at a frame rate of at least 20 frames per second.

Another embodiment is directed to a method for processing an array ofCMOS-fabricated sensors, each sensor comprising a chemically-sensitivefield effect transistor (chemFET). The method comprises: A) dicing asemiconductor wafer including the array to form at least one dicedportion including the array; and B) performing a forming gas anneal onthe at least one diced portion.

Another embodiment is directed to a method for manufacturing an array ofchemFETs. The method comprises: fabricating an array of chemFETs;depositing on the array a dielectric material; applying a forming gasanneal to the array before a dicing step; dicing the array; and applyinga forming gas anneal after the dicing step. The method may furthercomprise testing the semiconductor wafer between one or more depositionsteps.

Another embodiment is directed to a method for processing an array ofCMOS-fabricated sensors. Each sensor comprises a chemically-sensitivefield effect transistor (chemFET) having a chemically-sensitivepassivation layer of silicon nitride and/or silicon oxynitride depositedvia plasma enhanced chemical vapor deposition (PECVD). The methodcomprises depositing at least one additional passivation material on thechemically-sensitive passivation layer so as to reduce a porosity and/orincrease a density of the passivation layer.

Other aspects of the invention relate to methods for monitoring nucleicacid synthesis reactions, including but not limited to those integral tosequencing-by-synthesis methods. Thus, various aspects of the inventionprovide methods for monitoring nucleic acid synthesis reactions, methodsfor determining or monitoring nucleotide incorporation into a nucleicacid, methods for determining the presence or absence of nucleotideincorporation, methods for determining the number of incorporatednucleotides, and the like.

In one such aspect, the invention provides a method for sequencing anucleic acid comprising disposing (e.g., placing or positioning) aplurality of template nucleic acids into a plurality of reactionchambers, wherein the plurality of reaction chambers is in contact witha chemFET array comprising at least one chemFET for each reactionchamber, and wherein each of the template nucleic acids is hybridized toa sequencing primer (thereby forming a template/primer hybrid) and isbound to a polymerase, synthesizing a new nucleic acid strand (orextending the sequencing primer) by incorporating one or more knownnucleotide triphosphates (also referred to generically herein as dNTP)sequentially at the 3′ end of the sequencing primer, and detecting theincorporation of the one or more known nucleotide triphosphates by achange in voltage and/or current at the at least one chemFET. The array(and thus the plurality) of chemFETs may be comprised of at least two,at least three, at least four, at least five, at least six, at leastseven, at least eight, at least nine, at least ten, at least 100, atleast 200, at least 300, at least 400, at least 500, at least 1000, atleast 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, or more chemFET (orchemFET sensors, or sensors, as the terms are used interchangeablyherein). Similarly, the plurality of reaction chambers may be at leasttwo, at least three, at least four, at least five, at least six, atleast seven, at least eight, at least nine, at least ten, at least 100,at least 200, at least 300, at least 400, at least 500, at least 1000,at least 10⁴, at least 10⁵, at least 10⁶, at least 10⁷, or more reactionchambers.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemFET array, wherein at least onechemFET is in contact with each reaction chamber, and wherein each ofthe template nucleic acids is hybridized to a sequencing primer (therebyforming a template/primer hybrid) and is bound to a polymerase,synthesizing a new nucleic acid strand (or extending the sequencingprimer) by incorporating one or more known nucleotide triphosphatessequentially at the 3′ end of the sequencing primer, and detecting theincorporation of the one or more known nucleotide triphosphates by achange in voltage and/or current at the at least one chemFET, whereinthe chemFET array is any of the foregoing arrays.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemFET array comprising at least onechemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer (thereby forming atemplate/primer hybrid) and is bound to a polymerase, synthesizing a newnucleic acid strand (by extending the sequencing primer) byincorporating one or more known nucleotide triphosphates sequentially atthe 3′ end of the sequencing primer, and detecting the incorporation ofthe one or more known nucleotide triphosphates by a change in voltageand/or current at the at least one chemFET within the array, wherein acenter-to-center distance between adjacent reaction chambers (or“pitch”) is 1-10 μm.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemFET array comprising at least onechemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer (thereby forming atemplate/primer hybrid) and is bound to a polymerase, synthesizing a newnucleic acid strand (by extending the sequencing primer) byincorporating one or more known nucleotide triphosphates sequentially atthe 3′ end of the sequencing primer, and detecting the incorporation ofthe one or more known nucleotide triphosphates by the generation ofsequencing reaction byproduct, wherein (a) the chemFET array comprisesmore than 256 sensors, or (b) a center-to-center distance betweenadjacent reaction chambers is 1-10 μm. In an important embodiment, thesequencing reaction byproduct is PPi.

In yet another aspect, the invention provides a method for sequencing anucleic acid comprising fragmenting (or isolating, for example in thecontext of enriched exon isolation) a target nucleic acid to generate aplurality of fragmented (or isolated) nucleic acids, attaching each ofthe plurality of fragmented (or isolated) nucleic acids to individualbeads to generate a plurality of beads each attached to a singlefragmented (or isolated) nucleic acid, amplifying the number offragmented (or isolated) nucleic acids on each bead, delivering theplurality of beads attached to amplified fragmented (or isolated)nucleic acids to a chemFET array having a separate reaction chamber foreach sensor in the array, wherein only one bead is situated in eachreaction chamber, and performing simultaneous sequencing reactions inthe plurality of reaction chambers.

It is to be understood, as described in greater detail herein, that thenucleic acids to be sequenced may be derived from longer nucleic acidsthat are then subsequently fragmented (i.e., converted into shorternucleic acids) or they may be isolated at a length that is suitable tothe reactions contemplated herein and thus would not have to beshortened (or fragmented). It should therefore be understood that forevery aspect and limitation discussed herein that refers to the processof fragmenting a target nucleic acid, the method can also be carried outby isolating a target nucleic acid in the absence of fragmenting.

In still another aspect, the invention provides a method for sequencingnucleic acids comprising fragmenting a target nucleic acid to generate aplurality of fragmented nucleic acids, amplifying each fragmentednucleic acid separately in the presence of a bead and binding amplifiedcopies of the fragmented nucleic acid to the bead, thereby producing aplurality of beads each attached to a plurality of identical fragmentednucleic acids, delivering the plurality of beads each attached to aplurality of identical fragmented nucleic acids to a chemFET arrayhaving a separate reaction chamber for each chemFET sensor in the array,wherein only one bead is situated in each reaction chamber, andperforming simultaneous sequencing reactions in the plurality ofreaction chambers.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemFET array comprising at least onechemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer (thereby forming atemplate/primer hybrid) and is bound to a polymerase, synthesizing a newnucleic acid strand (by extending the sequencing primer) byincorporating one or more known nucleotide triphosphates sequentially atthe 3′ end of the sequencing primer, and detecting a change in the levelof a sequencing byproduct as an indicator of incorporation of the one ormore known nucleotide triphosphates.

The change in the level may an increase or a decrease in a levelrelative to a level prior to incorporation of the one or more knownnucleotide triphosphates. The change in the level may be read as achange in voltage and/or current at a chemFET sensor or a change in pH,but it is not so limited.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemFET array comprising at least onechemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer and is bound to apolymerase, synthesizing a new nucleic acid strand by incorporating oneor more known nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, directly detecting release of PPi as an indicator ofincorporation of the one or more known nucleotide triphosphates.

In another aspect, the invention provides a method for sequencingnucleic acids comprising fragmenting a template nucleic acid to generatea plurality of fragmented nucleic acids, attaching one strand from eachof the plurality of fragmented nucleic acids individually to beads togenerate a plurality of beads each having a single stranded fragmentednucleic acid attached thereto, delivering the plurality of beads havinga single stranded fragmented nucleic acid attached thereto to a chemFETarray having a separate reaction chamber for each sensor in the area,and wherein only one bead is situated in each reaction chamber, andperforming simultaneous sequencing reactions in the plurality ofchambers.

In one aspect, the invention provides a method for sequencing a nucleicacid comprising sequencing a plurality of identical template nucleicacids in a reaction chamber in contact with a chemFET, in an array whichcomprises at least 3 (and up to millions) of such assemblies of reactionchambers and chemFETs.

In another aspect, the invention provides a method for sequencing anucleic acid comprising detecting incorporation of one or more knownnucleotide triphosphates to a 3′ end of a sequencing primer hybridizedto a template nucleic acid in a reaction chamber in contact with achemFET of a chemFET array that comprises at least three chemFET.

In one aspect, the invention provides a method for sequencing a nucleicacid comprising fragmenting a target nucleic acid to generate aplurality of fragmented nucleic acids, individually amplifying one ormore of the plurality of fragmented nucleic acids, and sequencing theindividually amplified fragmented nucleic acids using a chemFET array.In one embodiment, the chemFET array comprises at least three chemFETs.In some embodiments, the chemFET array comprises at least 500 chemFETs,or at least 100,000 chemFETs. In some embodiments, the plurality offragmented nucleic acids is individually amplified using a water in oilemulsion amplification method.

In another aspect, the invention provides a method for sequencing anucleic acid comprising fragmenting a target nucleic acid to generate aplurality of fragmented nucleic acids, attaching each of the pluralityof fragmented nucleic acids to individual beads to generate a pluralityof beads each attached to a single fragmented nucleic acid, amplifyingthe fragmented nucleic acids on each bead resulting in a plurality ofidentical fragmented nucleic acids on each bead, delivering a pluralityof beads attached to fragmented nucleic acids to a chemFET array havinga separate reaction chamber for each sensor in the array, wherein onlyone bead is situated in each reaction chamber, and performing sequencingreactions simultaneously in the plurality of reaction chambers.

In another aspect, the invention provides a method for sequencing anucleic acid comprising fragmenting a target nucleic acid to generate aplurality of fragmented nucleic acids, individually amplifying one ormore of the plurality of fragmented nucleic acids, and sequencing theindividually amplified fragmented nucleic acids in a plurality ofreaction chambers having a center-to-center distance of about 1-10 μmusing a chemFET array. In various embodiments, the center-to-centerdistance is about 9 μm, about 5 μm, or about 2 μm.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a plurality of reaction chambers, wherein the plurality of reactionchambers is in contact with a chemFET array comprising at least onechemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer and is bound to apolymerase, synthesizing a new nucleic acid strand by incorporating oneor more known nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, and detecting incorporation of the one or more knownnucleotide triphosphates by a change in voltage at the at least onechemFET within the array in the absence of a detectable pH change.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of template nucleic acidsinto a reaction chamber, wherein the plurality of template nucleic acidsis attached to a single bead, each of the template nucleic acids ishybridized to a sequencing primer and is bound to a polymerase, and thereaction chamber is in contact with a chemFET, synthesizing a newnucleic acid strand by incorporating one or more known nucleotidetriphosphates sequentially at the 3′ end of the sequencing primer, anddetecting incorporation of the one or more known nucleotidetriphosphates by detection of a first and a second voltage pulse at thechemFET.

In still another aspect, the invention provides a method for sequencinga nucleic acid comprising disposing a plurality of template nucleicacids into a plurality of reaction chambers, wherein the plurality ofreaction chambers is in contact with a chemFET array comprising at leastone chemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer and is bound to apolymerase, synthesizing a new nucleic acid strand by incorporating oneor more known nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, and detecting incorporation of the one or more knownnucleotide triphosphates by non-enzymatically detecting releasedinorganic pyrophosphate. In one embodiment, the detecting step occurs inthe absence of a detectable pH change.

In still another aspect, the invention provides a method for sequencinga nucleic acid comprising disposing a plurality of template nucleicacids into a plurality of reaction chambers, wherein the plurality ofreaction chambers is in contact with a chemFET array comprising at leastone chemFET for each reaction chamber, and wherein each of the templatenucleic acids is hybridized to a sequencing primer and is bound to apolymerase, synthesizing a new nucleic acid strand by incorporating oneor more known nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, and detecting incorporation of the one or more knownnucleotide triphosphates by detecting released inorganic pyrophosphateand unincorporated nucleotide triphosphates.

In one embodiment, the released inorganic pyrophosphate is detected att₀ and unincorporated nucleotide triphosphates are detected at time t₁.In a related embodiment, a time difference of t₁−t₀ indicates a numberof known nucleotide triphosphates incorporated.

In another aspect, the invention provides a method for sequencing anucleic acid comprising contacting a template nucleic acid with asequencing primer and a polymerase for times and conditions sufficientto allow the template nucleic acid to bind to the sequencing primer forform a template/primer hybrid and to allow the polymerase to bind to thetemplate/primer hybrid, and synthesizing a new nucleic acid strand byincorporating nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, and detecting incorporation of the one or more knownnucleotide triphosphates by detecting released inorganic pyrophosphateand unincorporated nucleotide triphosphates.

In one embodiment, the released inorganic pyrophosphate is detected att₀ and unincorporated nucleotide triphosphates are detected at time t₁.In a related embodiment, the time difference between t₁ and t₀ (i.e.,t₁−t₀) indicates the number of known nucleotide triphosphatesincorporated.

In still another aspect, the invention provides a method for sequencinga nucleic acid comprising contacting a template nucleic acid with asequencing primer and a polymerase for times and conditions sufficientto allow the template nucleic acid to bind to the sequencing primer forform a template/primer hybrid and to allow the polymerase to bind to thetemplate/primer hybrid, and synthesizing a new nucleic acid strand (orextending sequencing primer) by incorporating one or more knownnucleotide triphosphates sequentially at the 3′ end of the sequencingprimer in a low ionic strength environment, and detecting incorporationof the one or more known nucleotide triphosphates by detection of one ormore voltage pulses at the chemFET.

Depending on the embodiment, the low ionic strength environment maycomprise less than 1 mM MgCl₂, less than 0.5 mM MgCl₂, less than 100 μMMgCl₂, or less than 50 μM MgCl₂. In other embodiments, the low ionicstrength environment may comprise less than 1 mM MnCl₂, less than 0.5 mMMnCl₂, less than 100 μM MnCl₂, or less than 50 μM MnCl₂. In still otherembodiments, the salt may be another Mg²⁺ or Mn²⁺ containing salt, or itmay be a Ca²⁺ or Co²⁺ containing salt, or it may be a combination of oneor more such salts.

In another aspect, the invention provides a method for determiningincorporation of a nucleotide triphosphate into a newly synthesizednucleic acid (or onto a primer such as a sequencing primer) comprisingcombining a nucleotide triphosphate, a template/primer hybrid, and apolymerase, in a solution in contact with a chemFET, and detectingvoltage pulses at the chemFET, wherein detection of a first and a secondvoltage pulse indicates incorporation of a nucleotide triphosphate andwherein detection of a first but not a second voltage pulse indicateslack of incorporation of a nucleotide triphosphate.

In one embodiment, the voltage pulses are detected at the chemFETindependent of a binding event at the passivation layer of the chemFET.

In another aspect, the invention provides a method for determiningincorporation of a nucleotide triphosphate into a newly synthesizednucleic acid (or onto a primer such as a sequencing primer) comprisingcombining a nucleotide triphosphate, a template/primer hybrid, and apolymerase, in a solution in contact with a chemFET, and detectingvoltage pulses at the chemFET independent of a binding event at thepassivation layer of the chemFET, wherein detection of a first and asecond voltage pulse indicates incorporation of at least one nucleotidetriphosphate.

In various embodiments of the some of the foregoing aspect, the firstvoltage pulse occurs at time t₀ and the second voltage pulse occurs attime t₁, and t₁−t₀ indicates the number of nucleotide triphosphatesincorporated. In various embodiments, the incorporated nucleotidetriphosphate is known. In various embodiments, the nucleotidetriphosphate is a plurality of identical nucleotide triphosphates, thetemplate/primer hybrid is a plurality of identical template/primerhybrids, and the polymerase is a plurality of identical polymerases.Alternatively, the polymerase may be a plurality of polymerases that arenot identical and that rather may be comprised of 2, 3, or more types ofpolymerase. In some instances, a mixture of two polymerases may be usedwith one having suitable processivity and the other having suitable rateof incorporation. The ratio of the different polymerases can vary andthe invention is not to be limited in this regard. Similarly thetemplate/primer hybrid may be a plurality of template/primer hybridswhich may not be identical to each other, provided that anytemplate/primer hybrids in a single reaction chamber or attached to asingle bead are identical to each other.

In still another aspect, the invention provides a method for sequencinga nucleic acid comprising disposing (e.g., placing or positioning) aplurality of template nucleic acids into a plurality of reactionchambers, wherein the plurality of reaction chambers is in contact witha chemFET array comprising at least one chemFET for each reactionchamber, and wherein each of the template nucleic acids is hybridized toa sequencing primer and is bound to a polymerase, synthesizing a newnucleic acid strand (or extending the sequencing primer) byincorporating one or more known nucleotide triphosphates sequentially atthe 3′ end of the sequencing primer, and detecting incorporation of theone or more known nucleotide triphosphates by detecting at the at leastone chemFET a first voltage pulse at time t₀ having height h₀ and asecond pulse at time t₁ having height h₁, wherein h₀ and h₁ are each atleast about 5 mV over baseline, and t₁−t₀ is at least 1 millisecond. Insome embodiments, t₁−t₀ is at least 5 millisecond, at least 10milliseconds, at least 20 milliseconds, at least 30 milliseconds, atleast 40 milliseconds, or at least 50 milliseconds.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of beads into a pluralityof reaction chambers, wherein each reaction chamber comprises a singlebead, each bead is attached to a plurality of identical template nucleicacids, each of the template nucleic acids is hybridized to a sequencingprimer and is bound to a polymerase, and wherein the plurality ofreaction chambers is in contact with a chemFET array comprising at leastone chemFET for each reaction chamber, synthesizing a new nucleic acidstrand (or extending the sequencing primer) by incorporating one or moreknown nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, and detecting the incorporation of the one or moreknown nucleotide triphosphates by detecting a first and a second voltagepulse at least one chemFET within the array, wherein the chemFET arraycomprises at least three chemFET.

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of beads into a pluralityof reaction chambers, wherein each reaction chamber comprises a singlebead, each bead is attached to a plurality of identical template nucleicacids, each of the template nucleic acids is hybridized to a sequencingprimer and is bound to a polymerase, and wherein the plurality ofreaction chambers is in contact with a chemical-sensitive field effecttransistor (chemFET) array comprising at least one chemFET for eachreaction chamber, initiating synthesis of a new nucleic acid strand (orextension of the sequencing primer) by introducing a plurality of knownidentical nucleotide triphosphates into each reaction chamber,synthesizing a new nucleic acid strand by incorporating one or moreknown nucleotide triphosphates sequentially at the 3′ end of thesequencing primer, and detecting the incorporation of the one or moreknown nucleotide triphosphates by detecting a first and a second voltagepulse at least one chemFET within the array.

In one embodiment, each reaction chamber comprises adenosinetriphosphate prior to introducing a plurality of known triphosphatesinto each reaction chamber.

In another aspect, the invention provides a method for sequencing anucleic acid comprising (a) disposing a plurality of beads into aplurality of reaction chambers, each reaction chamber comprising asingle bead, each bead attached to a plurality of identical templatenucleic acids, each of the template nucleic acids hybridized to asequencing primer and bound to a polymerase, and each reaction chamberin contact with at least one chemFET, (b) introducing a plurality ofknown identical nucleotide triphosphates into each reaction chamber, (c)detecting sequential incorporation at the 3′ end of the sequencingprimer of one or more nucleotide triphosphates if complementary tocorresponding nucleotides in the template nucleic acid, (d) washingunincorporated nucleotide triphosphates from the reaction chambers, and(e) repeating steps (b) through (d) in the same reaction chamber using adifferent plurality of known nucleotide triphosphates.

In one embodiment, sequential incorporation of one or more nucleotidetriphosphates is detected by a first and a second voltage pulse at thechemFET. In some embodiments, step (e) comprises repeating steps (b)through (d) by separately introducing each different plurality of knownnucleotide triphosphates into each reaction chamber

In another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of beads into a pluralityof reaction chambers, wherein each reaction chamber comprises a singlebead, each bead is attached to a plurality of identical template nucleicacids, each of the template nucleic acids is hybridized to a sequencingprimer and is bound to a polymerase, and wherein the each of theplurality of reaction chambers is in contact with at least one chemFETin a chemFET array, initiating synthesis of a new nucleic acid strand byintroducing a divalent cation into each reaction chamber, synthesizing anew nucleic acid strand by incorporating one or more known nucleotidetriphosphates sequentially at the 3′ end of the sequencing primer, anddetecting the incorporation of the one or more known nucleotidetriphosphates by detecting a first and a second voltage pulse at leastone chemFET within the array.

In one embodiment, the divalent cation is Mg²⁺. In another embodiment,the divalent cation is Mn²⁺. In still a further embodiment, the divalentcation is a mixture of Mg²⁺ and Mn²⁺. Depending on the embodiment, thedivalent cation is at a concentration of less than 1 mM, or less than100 μM, or about 50 μM.

In yet another aspect, the invention provides a method for sequencing anucleic acid comprising disposing a plurality of identical templatenucleic acids into a reaction chamber, wherein the reaction chamber isin contact with a chemFET, and wherein each of the template nucleicacids is hybridized to a sequencing primer and is bound to a polymerase,incorporating one or more known nucleotide triphosphates sequentially atthe 3′ end of the sequencing primer, and detecting the incorporation ofthe one or more known nucleotide triphosphates by a voltage pulse at thechemFET, wherein the incorporation of 10-1000 nucleotide triphosphatesis detected.

Various embodiments may be embraced in the various foregoing aspects ofthe invention and these are recited below once for convenience andbrevity.

It is to be understood that although various of the foregoing aspectsand embodiments of the invention recite hybridization (or binding) of asequencing primer to a template, the invention also contemplates the useof sequencing primers and/or template nucleic acids that hybridize tothemselves (i.e., intramolecularly) thereby giving rise to free 3′ endsonto which nucleotide triphosphates may be incorporated. Such templates,referred to herein as self-priming templates, may be used in any of theforegoing methods.

Similarly, the invention equally contemplates the use of double strandedtemplates that are engineered to have particular sequences at their freeends that can be acted upon by nicking enzymes such as DNA nickase. Inthis way, the polymerase incorporates nucleotide triphosphates at thenicked site. In these instances, there is no requirement for a separatesequencing primer.

In some embodiments, the incorporation of at least 10, at least 20, atleast 30, at least 40, at least 50, at least 60, at least 70, at least80, at least 90, or at least 100 nucleotide triphosphates is detected.In other embodiments, the incorporation of 100-1000 nucleotidetriphosphates is detected. In still other some embodiments, theincorporation of 250-750 nucleotide triphosphates is detected.

In some embodiments, the reaction chamber comprises a plurality ofpacking beads. In some embodiments, the detecting step occurs in thepresence of a plurality of packing beads.

In some embodiments, the reaction chamber comprises a solublenon-nucleic acid polymer. In some embodiments, the detecting step occursin the presence of a soluble non-nucleic acid polymer. In someembodiments, the soluble non-nucleic acid polymer is polyethyleneglycol, or PEA, or a dextran, or an acrylamide, or a cellulose (e.g.,methyl cellulose). In some embodiments, the non-nucleic acid polymersuch as polyethylene glycol is attached to the single bead. In someembodiments, the non-nucleic acid polymer is attached to one or more (orall) sides of a reaction chamber, except in some instances the bottom ofthe reaction chamber which is the FET surface In some embodiments, thenon-nucleic acid polymer is biotinylated such as but not limited tobiotinylated polyethylene glycol.

In some embodiments, the method is carried out at a pH of about 7-9, orat about 8.5 to 9.5, or at about 9.

In some embodiments, the synthesizing and/or detecting step is carriedout in a weak buffer. In some embodiments, the weak buffer comprisesTris-HCl, boric acid or borate buffer, acetate, morpholine, citric acid,carbonic acid, or phosphoric acid as a buffering agent.

In some embodiments, the synthesizing and/or detecting step is carriedout in about 1 mM Tris-HCl. In some embodiments, the synthesizing and/ordetecting step is carried out in less than 1 mM Tris-HCl. In someembodiments, the synthesizing and/or detecting step is carried out inabout 0.9 mM Tris-HCl, about 0.8 mM Tris-HCl, about 0.7 mM Tris-HCl,about 0.6 mM Tris-HCl, about 0.5 mM Tris-HCl, about 0.4 mM Tris-HCl,about 0.3 mM Tris-HCl, or about 0.2 mM Tris-HCl.

In some embodiments, the synthesizing and/or detecting step is carriedout in about 1 mM borate buffer. In some embodiments, the synthesizingand/or detecting step is carried out in less than 1 mM borate buffer. Insome embodiments, the synthesizing and/or detecting step is carried outin about 0.9 mM borate buffer, about 0.8 mM borate buffer, about 0.7 mMborate buffer, about 0.6 mM borate buffer, about 0.5 mM borate buffer,about 0.4 mM borate buffer, about 0.3 mM borate buffer, or about 0.2 mMborate buffer.

In some embodiments, the detection step occurs in the absence of adetectable pH change. In some embodiments, the detection step is carriedout in an environment of constant pH. In some embodiment, the chemFET isrelatively pH insensitive. In some embodiments, the synthesizing and/ordetecting step is carried out in about 0.5 mM Tris-HCl.

In some embodiments, the synthesizing and/or detecting step is carriedout in a low ionic strength environment. In some embodiments,synthesizing and/or detecting step is carried out in a low ionicstrength environment that comprises less than 1 mM MgCl₂ or MnCl₂, lessthan 0.5 mM MgCl₂ or MnCl₂, or less than 100 μM MgCl₂ or MnCl₂. In someembodiments, the synthesizing and/or detecting step is carried out in alow ionic strength environment that comprises about 100 μM MgCl₂ orMnCl₂, about 75 μM MgCl₂ or MnCl₂, about 50 μM MgCl₂ or MnCl₂, about 40μM MgCl₂ or MnCl₂, about 30 μM MgCl₂ or MnCl₂, about 20 μM MgCl₂ orMnCl₂, or about 10 μM MgCl₂ or MnCl₂. It is to be understood that theinvention contemplates similarly amounts of other divalent cations andit is therefore not limited to MgCl₂ or MnCl₂ only. Similarly it shouldbe understood that the invention contemplates the use of two or moredivalent cations (and/or their corresponding salts) provided the totalion concentration is at the levels stated above.

In some embodiments, the synthesizing and/or detecting step is carriedout in about 0.5 mM TRIS and about 50 μM MgCl₂ or MnCl₂.

In various embodiments, the nucleotide triphosphates are unblocked. Asused herein, an unblocked nucleotide triphosphate is a nucleotidetriphosphate with an unmodified end that can be incorporated into anucleic acid (at its 3′ end) and once it is incorporated can be attachedto the following nucleotide triphosphate being incorporated. BlockeddNTP in contrast either cannot be added to a nucleic acid or theirincorporation into a nucleic acid prevents any further nucleotideincorporation. In various embodiments, the nucleotide triphosphates aredeoxynucleotide triphosphates (dNTPs).

In various embodiments, the chemFET comprises a silicon nitridepassivation layer. In some embodiments, the chemFET comprises apassivation layer attached to inorganic pyrophosphate (PPi) receptors.In some embodiments, the chemFET comprises a passivation layer that isnot bound to a nucleic acid.

In some embodiments, each reaction chamber is in contact with a singlechemFET.

In some embodiments, the reaction chamber has a volume of equal to orless than about 1 picoliter (pL), including less than 0.5 pL, less than0.1 pL, less than 0.05 pL, less than 0.01 pL, less than 0.005 pL. Insome embodiment, the reaction chambers are separated by acenter-to-center spacing of 1-10 μm. In some embodiments, the reactionchambers are separated by a center-to-center spacing of about 9 μm,about 5 microns, or about 2 microns. The reaction chambers may have asquare cross section, for example at their base or bottom. Examplesinclude an 8 μm by 8 μm cross section, a 4 μm by 4 μm cross section, ora 1.5 μm by 1.5 μm cross section. Alternatively, they may have arectangular cross section, for example at their base or bottom. Examplesinclude an 8 μm by 12 μm cross section, a 4 μm by 6 μm cross section, ora 1.5 μm by 2.25 μm cross section.

In some embodiments, the nucleotide triphosphates are pre-soaked in Mg²⁺(e.g., in the presence of MgCl₂) or Mn²⁺ (for example in the presence ofMnCl₂). In some embodiments, the polymerase is pre-soaked in Mg²⁺ (e.g.,in the presence of MgCl₂) or Mn²⁺ (for example in the presence ofMnCl₂).

In some embodiments, the method is carried out in a reaction chambercomprising a single capture bead, wherein a ratio of reaction chamberwidth to single capture bead diameter is at least 0.7, at least 0.8, orat least 0.9.

In some embodiments, the polymerase is free in solution. In someembodiments, the polymerase is immobilized to a bead. In someembodiments, the polymerase is immobilized to a capture bead. In someembodiments, the template nucleic acids are attached to capture beads.

In some embodiments, inorganic pyrophosphate (PPi) is not significantlyhydrolyzed prior to detecting nucleotide incorporation. In someembodiments, inorganic pyrophosphate (PPi) is not significantlyhydrolyzed prior to detecting a first voltage pulse.

In one aspect, the invention provides an apparatus comprising a chemFETarray comprising a plurality of chemFET sensors each having a PPireceptor disposed on its surface, wherein the array comprises at leastthree chemFET sensors.

In one aspect, the invention provides an apparatus comprising a chemFETarray comprising a plurality of chemFET sensors each having a PPireceptor disposed on its surface, wherein adjacent chemFET sensors inthe array are separated by a center-to-center distance of about lessthan about 10 μm (e.g., using 0.18 μm CMOS fabrication, the pitch may beabout 2.8 μm).

In another aspect, the invention provides an apparatus comprising achemical-sensitive field effect transistor (chemFET) having disposed onits surface a PPi selective receptor. The PPi receptor may beimmobilized to the chemFET surface.

In still another aspect, the invention provides an apparatus comprisinga chemical-sensitive field effect transistor (chemFET) array comprisinga plurality of chemFET sensors each having a PPi receptor disposed onits surface, wherein the plurality is a subset of the chemFET sensors inthe array. In a related embodiment, the array comprises at least threechemFET sensors. The subset may represent 10%, 25%, 33%, 50%, 75% ormore of the sensors in the array. The PPi selective receptor may beimmobilized to the surface of each chemFET.

In some embodiments, the center-to-center distance between adjacentreaction chambers is about 1-9 μm, or about 2-9 μm, about 1 μm, about 2μm, about 3 μm, about 4 μm, about 5 μm, about 6 μm, about 7 μm, about 8μm, or about 9 μm. In some embodiments, the chemFET array comprises morethan 256 sensors (and optionally more than 256 corresponding reactionchambers (or wells)), more than 300 sensors (and optionally more than300 corresponding reaction chambers), more than 400 sensors (andoptionally more than 400 corresponding reaction chambers), more than 500sensors (and optionally more than 500 corresponding reaction chambers),more than 600 sensors (and optionally more than 600 correspondingreaction chambers), more than 700 sensors (and optionally more than 700corresponding reaction chambers), more than 800 sensors (and optionallymore than 800 corresponding reaction chambers), more than 900 sensors(and optionally more than 900 corresponding reaction chambers), morethan 10³ sensors (and optionally more than 10³ corresponding reactionchambers), more than 10⁴ sensors (and optionally more than 10⁴corresponding reaction chambers), more than 10⁵ sensors (and optionallymore than 10⁵ corresponding reaction chambers), or more than 10⁶ sensors(and optionally more than 10⁶ corresponding reaction chambers). In someembodiments, the chemFET array comprises at least 512 rows and at least512 columns of sensors.

In some embodiments, the sequencing byproduct is PPi. In someembodiments, PPi is measured directly. In related embodiments, PPi isdetected by its binding to a PPi selective receptor immobilized on thesurface of a chemFET sensor. In another embodiment, PPi is detected inthe absence of a PPi selective receptor. In other embodiments, PPi isdetected in a pH independent or insensitive environment.

In some embodiments, the sequencing reaction byproduct is hydrogen ion.In some embodiments, the sequencing reaction byproduct is Pi. In stillother embodiments, the chemFET detects changes in any combination of thebyproducts, optionally in combination with other parameters, asdescribed herein.

In some embodiments, the PPi selective receptor is Compound 1, Compound2, Compound 3, Compound 4, Compound 5, Compound 6, Compound 7, Compound8, Compound 9 or Compound 10 as shown in FIG. 11B. In some embodiments,the chemFET is present in an array of chemFETs, each of which hasdisposed, including immobilized, on its surface a PPi selectivereceptor. In some embodiments, identical PPi selective receptors aredisposed on each chemFET of the array. In some embodiments, the arraycomprises more than 256 sensors. In some embodiments, the arraycomprises at least 512 rows and at least 512 columns of sensors. In someembodiments, the chemFET is located at a bottom of a reaction chamber.

In another aspect, the invention provides an apparatus comprising achemFET array having disposed on its surface a biological array or achemical array.

The biological array may be a nucleic acid array, a protein arrayincluding but not limited to an enzyme array, an antibody array and anantibody fragment array, a cell array, and the like. The chemical arraymay be an organic small molecule array, or an inorganic molecule array,but it is not so limited. The chemFET array may comprise at least 5, atleast 10, at least 10², at least 10³, at least 10⁴, at least 10⁵, atleast 10⁶, or more sensors. The biological or chemical array may bearranged into a plurality of “cells” or spatially defined regions, andeach of these regions is situated over a different sensor in the chemFETarray, in some embodiments.

In yet another aspect, the invention provides a method for detecting anucleic acid comprising contacting a nucleic acid array disposed on achemFET array with a sample, and detecting binding of a nucleic acidfrom the sample to one or more regions on the nucleic acid array.

In another aspect, the invention provides a method for detecting aprotein comprising contacting a protein array disposed on a chemFETarray with a sample, and detecting binding of a protein from the sampleto one or more regions on the protein array.

In yet another aspect, the invention provides a method for detecting anucleic acid comprising contacting a protein array disposed on a chemFETarray with a sample, and detecting binding of a nucleic acid from thesample to one or more regions on the protein array.

In another aspect, the invention provides a method for detecting anantigen comprising contacting an antibody array disposed on a chemFETarray with a sample, and detecting binding of an antigen from the sampleto one or more regions on the antibody array.

In another aspect, the invention provides a method for detecting anenzyme substrate or inhibitor comprising contacting an enzyme arraydisposed on a chemFET array with a sample, and detecting binding of anentity from the sample to one or more regions on the enzyme array.

It should be appreciated that all combinations of the foregoing conceptsand additional concepts discussed in greater detail below (provided suchconcepts are not mutually inconsistent) are contemplated as being partof the inventive subject matter disclosed herein. In particular, allcombinations of claimed subject matter appearing at the end of thisdisclosure are contemplated as being part of the inventive subjectmatter disclosed herein. It should also be appreciated that terminologyexplicitly employed herein that also may appear in any disclosureincorporated by reference should be accorded a meaning most consistentwith the particular concepts disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

In the drawings, like reference characters generally refer to the sameparts throughout the different views. Also, the drawings are notnecessarily to scale, emphasis instead being placed upon generallyillustrating the various concepts discussed herein.

FIG. 1 illustrates a cross-section of a p-type (p-channel) ion-sensitivefield effect transistor (ISFET) fabricated using a conventional CMOSprocess.

FIG. 2 illustrates an electric circuit representation of the p-channelISFET shown in FIG. 1.

FIG. 2A illustrates an exemplary ISFET transient response to astep-change in ion concentration of an analyte.

FIG. 3 illustrates one column of a two-dimensional ISFET array based onthe ISFET shown in FIG. 1.

FIG. 4 illustrates a transmission gate including a p-channel MOSFET andan re-channel MOSFET that is employed in each pixel of the array columnshown in FIG. 3.

FIG. 5 is a diagram similar to FIG. 1, illustrating a widercross-section of a portion of a substrate corresponding to one pixel ofthe array column shown in FIG. 3, in which the ISFET is shown alongsidetwo n-channel MOSFETs also included in the pixel.

FIG. 6 is a diagram similar to FIG. 5, illustrating a cross-section ofanother portion of the substrate corresponding to one pixel of the arraycolumn shown in FIG. 3, in which the ISFET is shown alongside thep-channel MOSFET of the transmission gate shown in FIG. 4.

FIG. 7 illustrates an example of a complete two-dimensional ISFET pixelarray based on the column design of FIG. 3, together with accompanyingrow and column decoder circuitry and measurement readout circuitry.

FIG. 8 generally illustrates a nucleic acid processing system comprisinga large scale chemFET array, according to one inventive embodiment ofthe present disclosure.

FIG. 8A is a graph illustrating plots of ionized species concentrationin proximity of an analyte/passivation layer interface of a chemFET as afunction of time for an exemplary nucleic acid sequencing/synthesisreaction according to one embodiment of the present invention.

FIG. 8B is a graph illustrating multiple plots of chemFET output voltageas a function of time for exemplary nucleic acid sequencing/synthesisreactions similar to those profiled in FIG. 8A, according to oneembodiment of the present invention.

FIG. 9 illustrates one column of an chemFET array similar to that shownin FIG. 8, according to one inventive embodiment of the presentdisclosure.

FIG. 9A illustrates a circuit diagram for an exemplary amplifieremployed in the array column shown in FIG. 9.

FIG. 9B is a graph of amplifier bias vs. bandwidth, according to oneinventive embodiment of the present disclosure.

FIG. 10 illustrates a top view of a chip layout design for a pixel ofthe column of an chemFET array shown in FIG. 9, according to oneinventive embodiment of the present disclosure.

FIG. 10-1 illustrates a top view of a chip layout design for a clusterof four neighboring pixels of an chemFET array shown in FIG. 9,according to another inventive embodiment of the present disclosure.

FIG. 11A shows a composite cross-sectional view along the line I-I ofthe pixel shown in FIG. 10, including additional elements on the righthalf of FIG. 10 between the lines II-II and III-III, illustrating alayer-by-layer view of the pixel fabrication according to one inventiveembodiment of the present disclosure.

FIG. 11A-1 shows a composite cross-sectional view of multipleneighboring pixels, along the line I-I of one of the pixels shown inFIG. 10-1, including additional elements of the pixel between the linesII-II, illustrating a layer-by-layer view of pixel fabrication accordingto another inventive embodiment of the present disclosure.

FIGS. 11B(1)-(3) provide the chemical structures of ten PPi receptors(compounds 1 through 10).

FIG. 11C(1) is a schematic of a synthesis protocol for compound 7 fromFIG. 11B(3).

FIG. 11C(2) is a schematic of a synthesis protocol for compound 8 fromFIG. 11B(3).

FIG. 11C(3) is a schematic of a synthesis protocol for compound 9 fromFIG. 11B(3).

FIGS. 11D(1) and (2) are schematics illustrating a variety ofchemistries that can be applied to the passivation layer in order tobind molecular recognition compounds (such as but not limited to PPireceptors).

FIG. 11E is a schematic of attachment of compound 7 from FIG. 11B(3) toa metal oxide surface.

FIGS. 12A through 12L provide top views of each of the fabricationlayers shown in FIG. 11A, according to one inventive embodiment of thepresent disclosure.

FIGS. 12-1A through 12-1L provide top views of each of the fabricationlayers shown in FIG. 11A-1, according to another inventive embodiment ofthe present disclosure.

FIG. 13 illustrates a block diagram of an exemplary CMOS IC chipimplementation of an chemFET sensor array similar to that shown in FIG.8, based on the column and pixel designs shown in FIGS. 9-12, accordingto one inventive embodiment of the present disclosure.

FIG. 14 illustrates a row select shift register of the array shown inFIG. 13, according to one inventive embodiment of the presentdisclosure.

FIG. 15 illustrates one of two column select shift registers of thearray shown in FIG. 13, according to one inventive embodiment of thepresent disclosure.

FIG. 16 illustrates one of two output drivers of the array shown in FIG.13, according to one inventive embodiment of the present disclosure.

FIG. 17 illustrates a block diagram of the chemFET sensor array of FIG.13 coupled to an array controller, according to one inventive embodimentof the present disclosure.

FIG. 18 illustrates an exemplary timing diagram for various signalsprovided by the array controller of FIG. 17, according to one inventiveembodiment of the present disclosure.

FIG. 18A illustrates another exemplary timing diagram for varioussignals provided by the array controller of FIG. 17, according to oneinventive embodiment of the present disclosure.

FIG. 18B shows a flow chart illustrating an exemplary method forprocessing and correction of array data acquired at high acquisitionrates, according to one inventive embodiment of the present disclosure.

FIGS. 18C and 18D illustrate exemplary pixel voltages showingpixel-to-pixel transitions in a given array output signal, according toone embodiment of the present disclosure.

FIGS. 19-20 illustrate block diagrams of alternative CMOS IC chipimplementations of chemFET sensor arrays, according to other inventiveembodiments of the present disclosure.

FIG. 20A illustrates a top view of a chip layout design for a pixel ofthe chemFET array shown in FIG. 20, according to another inventiveembodiment of the present disclosure.

FIGS. 21-23 illustrate block diagrams of additional alternative CMOS ICchip implementations of chemFET sensor arrays, according to otherinventive embodiments of the present disclosure.

FIG. 24 illustrates the pixel design of FIG. 9 implemented with ann-channel chemFET and accompanying n-channel MOSFETs, according toanother inventive embodiment of the present disclosure.

FIGS. 25-27 illustrate alternative pixel designs and associated columncircuitry for chemFET arrays according to other inventive embodiments ofthe present disclosure.

FIGS. 28A and 28B are isometric illustrations of portions of microwellarrays as employed herein, showing round wells and rectangular wells, toassist three-dimensional visualization of the array structures.

FIG. 29 is a diagrammatic depiction of a top view of one corner (i.e.,the lower left corner) of the layout of a chip showing an array ofindividual ISFET sensors on a CMOS die.

FIG. 30 is an illustration of an example of a layout for a portion of a(typically chromium) mask for a one-sensor-per-well embodiment of theabove-described sensor array, corresponding to the portion of the dieshown in FIG. 29.

FIG. 31 is a corresponding layout for a mask for a 4-sensors-per-wellembodiment.

FIG. 32 is an illustration of a second mask used to mask an area whichsurrounds the array, to build a collar or wall (or basin, using thatterm in the geological sense) of resist which surrounds the active arrayof sensors on a substrate, as shown in FIG. 33A.

FIG. 33 is an illustration of the resulting basin.

FIG. 33A is an illustration of a three-layer PCM process for making themicrowell array.

FIG. 33B is a diagrammatic cross-section of a microwell with a “bump”feature etched into the bottom.

FIG. 33B-1 is an image from a scanning electron microscope showing incross-section a portion of an array architecture as taught herein, withmicrowells formed in a layer of silicon dioxide over ISFETs.

FIG. 33B-2 is a diagrammatic illustration of a microwell incross-section, the microwell being produced as taught herein and havingsloped sides, and showing how a bead of a correspondingly appropriatediameter larger than that of the well bottom can be spaced from the wellbottom by interference with the well sidewalls.

FIG. 33B-3 is another diagrammatic illustration of such a microwell withbeads of different diameters shown, and indicating optional use ofpacking beads below the nucleic acid-carrying bead such as aDNA-carrying bead.

FIGS. 34-37 diagrammatically illustrate a first example of a suitableexperiment apparatus incorporating a fluidic interface with the sensorarray, with FIG. 35 providing a cross-section through the FIG. 34apparatus along section line 35-35′ and FIG. 36 expanding part of FIG.35, in perspective, and FIG. 37 further expanding a portion of thestructure to make the fluid flow more visible.

FIG. 38 is a diagrammatic illustration of a substrate with an etchedphotoresist layer beginning the formation of an example flow cell of acertain configuration.

FIGS. 39-41 are diagrams of masks suitable for producing a firstconfiguration of flow cell consistent with FIG. 38.

FIGS. 42-54 (but not including FIGS. 42A-42L) and 57-58 are pairs ofpartly isometric, sectional views of example apparatus and enlargements,showing ways of introducing a reference electrode into, and forming, aflow cell and flow chamber, using materials such as plastic and PDMS.

FIG. 42A is an illustration of a possible cross-sectional configurationof a non-rectangular flow chamber antechamber (diffuser section) for useto promote laminar flow into a flow cell as used in the arrangementsshown herein;

FIGS. 42B-42F are diagrammatic illustrations of examples of flow cellstructures for unifying fluid flow.

FIG. 42F1 is a diagrammatic illustration of an example of a ceilingbaffle arrangement for a flow cell in which fluid is introduced at onecorner of the chip and exits at a diagonal corner, the bafflearrangement facilitating a desired fluid flow across the array.

FIGS. 42F2-42F8 comprise a set of illustrations of an exemplary flowcell member that may be manufactured by injection molding and mayincorporate baffles to facilitate fluid flow, as well as a metalizedsurface for serving as a reference electrode, including an illustrationof said member mounted to a sensor array package over a sensor array, toform a flow chamber thereover.

FIGS. 42G and 42H are diagrammatic illustrations of alternativeembodiments of flow cells in which fluid flow is introduced to themiddle of the chip assembly.

FIGS. 42I and 42J are cross-sectional illustrations of the type of flowcell embodiments shown in FIGS. 42G and 42H, mounted on a chip assembly;

FIGS. 42K and 42L are diagrammatic illustrations of flow cells in whichthe fluid is introduced at a corner of the chip assembly.

FIG. 42M is a diagrammatic illustration of fluid flow from one corner ofan array on a chip assembly to an opposite corner, in apparatus such asthat depicted in FIGS. 42K and 42L.

FIGS. 55 and 56 are schematic, cross-sectional views of two-layer glass(or plastic) arrangements for manufacturing fluidic apparatus formounting onto a chip for use as taught herein.

FIGS. 57 and 58 are schematic embodiments of a fluidic assembly.

FIGS. 59A-59C are illustrations of the pieces for two examples oftwo-piece injection molded parts for forming a flow cell.

FIG. 60 is a schematic illustration, in cross-section, for introducing astainless steel capillary tube as an electrode, into a downstream portof a flow cell such as the flow cells of FIGS. 59A-59C, or other flowcells.

FIG. 61 is a schematic illustrating the incorporation of a dNTP into asynthesized nucleic acid strand with concomitant release of inorganicpyrophosphate (PPi).

FIGS. 61A and B are data capture images of microwell arrays followingbead deposition. The white spots are beads. FIG. 61A is an opticalmicroscope image and FIG. 61B is an image captured using the chemFETsensor underlying the microwell array.

FIGS. 62-70 illustrate bead loading into the microfluidic arrays of theinvention.

FIG. 71 illustrates an exemplary sequencing process.

FIGS. 72A-C provide a schematic of a polymerase assay (A), and extensiondata for various polymerases.

FIG. 73 provides data relating to polymerase affinity in low ionicstrength.

FIGS. 74A-C provide data relating to polymerase activity as a functionof time.

FIG. 75A-B illustrate the well and sensor system used to assay primerextension (A) and the results of such extension assays (B).

DETAILED DESCRIPTION

Following below are more detailed descriptions of various conceptsrelated to, and embodiments of, inventive methods and apparatus relatingto large scale chemFET arrays for analyte detection and/or measurement.It should be appreciated that various concepts introduced above anddiscussed in greater detail below may be implemented in any of numerousways, as the disclosed concepts are not limited to any particular mannerof implementation. Examples of specific implementations and applicationsare provided primarily for illustrative purposes.

Various inventive embodiments according to the present disclosure aredirected at least in part to a semiconductor-based/microfluidic hybridsystem that combines the power of microelectronics with thebiocompatibility of a microfluidic system. In some examples below, themicroelectronics portion of the hybrid system is implemented in CMOStechnology for purposes of illustration. It should be appreciated,however, that the disclosure is not intended to be limiting in thisrespect, as other semiconductor-based technologies may be utilized toimplement various aspects of the microelectronics portion of the systemsdiscussed herein.

One embodiment disclosed herein is directed to a large sensor array(e.g., a two-dimensional array) of chemically-sensitive field effecttransistors (chemFETs), wherein the individual chemFET sensor elementsor “pixels” of the array are configured to detect analyte presence (orabsence), analyte levels (or amounts), and/or analyte concentration inan unmanipulated sample, or as a result of chemical and/or biologicalprocesses (chemical reactions, cell cultures, neural activity, nucleicacid sequencing processes, etc.) occurring in proximity to the array.Examples of chemFETs contemplated by various embodiments discussed ingreater detail below include, but are not limited to, ion-sensitivefield effect transistors (ISFETs) and enzyme-sensitive field effecttransistors (ENFETs). In one exemplary implementation, one or moremicrofluidic structures is/are fabricated above the chemFET sensor arrayto provide for containment and/or confinement of a biological orchemical reaction in which an analyte of interest may be produced orconsumed, as the case may be. For example, in one implementation, themicrofluidic structure(s) may be configured as one or more “wells”(e.g., small reaction chambers or “reaction wells”) disposed above oneor more sensors of the array, such that the one or more sensors overwhich a given well is disposed detect and measure analyte presence,level, and/or concentration in the given well.

In another exemplary implementation, the invention encompasses a systemfor high-throughput sequencing comprising at least one two-dimensionalarray of reaction chambers, wherein each reaction chamber is coupled toa chemically-sensitive field effect transistor (“chemFET”) and eachreaction chamber is no greater than 10 μm³ (i.e., 1 pL) in volume.Preferably, each reaction chamber is no greater than 0.34 pL, and morepreferably no greater than 0.096 pL or even 0.012 pL in volume. Areaction chamber can optionally be 2², 3², 4², 5², 6², 7², 8², 9², or10² square microns in cross-sectional area at the top. Preferably, thearray has at least 100, 1,000, 10,000, 100,000, or 1,000,000 reactionchambers. The reaction chambers may be capacitively coupled to thechemFETs, and preferably are capacitively coupled to the chemFETs.

In some embodiments, such a chemFET array/microfluidics hybrid structuremay be used to analyze solution(s)/material(s) of interest containingnucleic acids. For example, such structures may be employed to monitorsequencing of nucleic acids. Sequencing of nucleic acids may beperformed to determine partial or complete nucleotide sequence of anucleic acid, to detect the presence and in some instances nature of asingle nucleotide polymorphism in a nucleic acid, to determine whattherapeutic regimen will be most effective to treat a subject having aparticular condition as can be determined by the subject's geneticmake-up, to determine and compare nucleic acid expression profiles oftwo or more states (e.g., comparing expression profiles of diseased andnormal tissue, or comparing expression profiles of untreated tissue andtissue treated with drug, enzymes, radiation or chemical treatment), tohaplotype a sample (e.g., comparing genes or variations in genes on eachof the two alleles present in a human subject), to karyotype a sample(e.g., analyzing chromosomal make-up of a cell or a tissue such as anembryo, to detect gross chromosomal or other genomic abnormalities), andto genotype (e.g., analyzing one or more genetic loci to determine forexample carrier status and/or species-genus relationships).

The systems described herein can be utilized to sequence the nucleicacids of an entire genome, or any portion thereof. Genomes that can besequenced include mammalian genomes, and preferably human genomes. Thus,in one exemplary embodiment, the invention encompasses a method forsequencing a genome or part thereof comprising: delivering fragmentednucleic acids from the genome or part thereof to a system forhigh-throughput sequencing comprising at least one two-dimensional arrayof reaction chambers, wherein each reaction chamber is coupled to achemically-sensitive field effect transistor (“chemFET”) and eachreaction chamber is no greater than 1 pL in volume; and detecting asequencing reaction in at least one of the reaction chambers via asignal from the chemFET of the reaction chamber.

In an alternative exemplary embodiment, the method comprises: deliveringfragmented nucleic acids from the genome or part thereof to a sequencingapparatus comprising a two-dimensional array of reaction chambers,wherein each of the reaction chambers is disposed in a sensingrelationship with an associated chemFET; and detecting a sequencingreaction in at least one of the reaction chambers via a signal from theassociated chemFET.

Preferably, the sequencing reaction is performed by delivering a firstdeoxyribonucleotide triphosphate (“dNTP”) to each of the reactionchambers. Preferably, the delivering step comprises delivering the firstdNTP at substantially the same time to each of the reaction chambers.Once the first dNTP is delivered to the reaction chambers, an enzyme istypically delivered to the reaction chambers to degrade any unused dNTP,followed by washing to remove substantially all of the enzyme from thereaction chambers. Preferably, the enzyme also degrades PPi. The washingcan also remove any degraded dNTP or degraded PPi.

The nucleic acids to be sequenced can be naturally or non-naturallyoccurring nucleic acids, and preferably are naturally occurring nucleicacids. The nucleic acids can be obtained from any of several sourcesincluding, but not limited to, deoxyribonucleic acid (“DNA”) (e.g.,messenger DNA, complementary DNA, or nuclear DNA), ribonucleic acid(“RNA”) (e.g., micro RNA, transfer RNA, messenger RNA, or smallinterfering RNA), or peptides. When the source is DNA, the DNA can beobtained from any bodily fluid or tissue that contains DNA, including,but not limited to, blood, saliva, cerebrospinal fluid (“CSF”), skin,hair, urine, stool, and mucus. The starting amounts of nucleic acids tobe sequenced determine the minimum sample requirements. Considering thefollowing bead sizes, with an average of 450 bases in the single strand,with an average molecular weight of 325 g/mol per base we have thefollowing:

Bead Size (um) femto gram of DNA 0.2 0.124 0.3 0.279 0.7 1.52 1.05 3.422.8 24.3 5.9 108

Given the number of beads and microwells contemplated for use in anarray, it will thus be apparent that a sample taken from a subject to betested need only be on the order of 3 μg.

Preferably, at least 10⁶ base pairs are sequenced per hour, morepreferably at least 10⁷ base pairs are sequenced per hour, even morepreferably at least 10⁸ base pairs are sequenced per hour, even morepreferably at least 10⁹ base pairs are sequenced per hour, and mostpreferably at least 10¹⁰ base pairs are sequenced per hour using theabove-described method. Thus, the method may be used to sequence anentire human genome within about 24 hours, more preferably within about20 hours, even more preferably within about 15 hours, even morepreferably within about 10 hours, even more preferably within about 5hours, and most preferably within about 1 hour.

The systems described herein can be utilized to sequence an entiregenome of an organism from about 3 μg of DNA or less. In one embodiment,the invention encompasses a method for sequencing an entire genome of anorganism comprising delivering about 3 μg of DNA or less from theorganism to an array of chemically sensitive field effect transistorsand determining a sequence of the genome. In another embodiment, theinvention encompasses a device adapted for sequencing a complete humangenome from about 1 ng of DNA or less without the use of optics orlabels. Typically, the device comprises an array of chemFETs and/or anarray of microfluidic reaction chambers and/or a semiconductor materialcoupled to a dielectric material.

The above-described sequencing can be performed in a reaction mixturehaving an ionic strength of up to 500 μM, 400 μM, or 300 μM. The ionicstrength, I, of a solution is a function of the concentration of allions present in a solution.

$I_{c} = {\frac{1}{2}{\sum\limits_{B = 1}^{n}{c_{B}z_{B}^{2}}}}$where c_(B) is the molar concentration of ion B (mol dm⁻³), z_(B) is thecharge number of that ion, and the sum is taken over all ions in thesolution. The Mg⁺² or Mn²⁺ concentration may be 1000 μM, 500 μM, 200 μM,100 μM, 50 μM, 10 μM, 5 μM, or even 1 μM.

The above-described method may be automated such that the sequencingreactions are performed via robotics. In addition, the sequencinginformation obtained via the signal from the chemFET of the reactionchamber may be provided to a personal computer, a personal digitalassistant, a cellular phone, a video game system, or a television sothat a user can monitor the progress of the sequencing reactionsremotely. This process is illustrated, for example, in FIG. 71.

The systems described herein can also be used to aid in theidentification and treatment of disease. For example, the system can beused for identifying a sequence associated with a particular disease orfor identifying a sequence associated with a positive response to aparticular active ingredient.

In one embodiment, the invention encompasses a method for identifying asequence associated with a condition comprising: delivering nucleicacids from a plurality of subjects having the condition to a sequencingapparatus comprising a two-dimensional array of reaction chambers,wherein each of the reaction chambers is capacitively coupled to achemFET; determining sequences of the nucleic acids from signal fromsaid chemFETs; and identifying a common sequence between the DNA fromthe plurality of subjects. Preferably, the subject is a mammal, and morepreferably a human. Preferably, the condition is cancer, animmunosuppressant condition, a neurological condition, or a viralinfection.

In another embodiment, the invention encompasses a method foridentifying a sequence associated with a positive response to aparticular active agent, comprising: sequencing DNA from a plurality ofsubjects that have exhibited a positive response and from a plurality ofsubjects having a negative response to an active agent using one or moresequencing apparatuses, wherein each sequencing apparatus comprises anarray of chemFETs; and identifying a common DNA sequence in theplurality of subjects that have exhibited a positive response or fromthe subjects that have exhibited a negative response that is not presentin the other plurality of subjects. Preferably, the subject is a mammal,and more preferably a human.

It should be appreciated, however, that while some illustrative examplesof the concepts disclosed herein focus on nucleic acid processing, theinvention contemplates a broader application of these concepts and isnot intended to be limited to these examples.

FIG. 8 generally illustrates a nucleic acid processing system 1000comprising a large scale chemFET array, according to one inventiveembodiment of the present disclosure. An example of a nucleic acidprocessing system is a nucleic acid sequencing system. In the discussionthat follows, the chemFET sensors of the array are described forpurposes of illustration as ISFETs configured for sensitivity to staticand/or dynamic ion concentration, including but not limited to hydrogenion concentration and/or concentration of other ionic species involvedin nucleic acid processing. However, it should be appreciated that thepresent disclosure is not limited in this respect, and that in any ofthe embodiments discussed herein in which ISFETs are employed as anillustrative example, other types of chemFETs may be similarly employedin alternative embodiments, as discussed in further detail below.Similarly it should be appreciated that various aspects and embodimentsof the invention may employ ISFETs as sensors yet detect one or moreionic species that are not hydrogen ions.

The system 1000 includes a semiconductor/microfluidics hybrid structure300 comprising an ISFET sensor array 100 and a microfluidics flow cell200. In one aspect, the flow cell 200 may comprise a number of wells(not shown in FIG. 8) disposed above corresponding sensors of the ISFETarray 100. In another aspect, the flow cell 200 is configured tofacilitate the sequencing of one or more identical template nucleicacids disposed in the flow cell via the controlled and ordered admission(or introduction) to the flow cell of a number of sequencing reagents272 (e.g., bases dATP, dCTP, dGTP, dTTP, generically referred to hereinas dNTP, divalent cations such as but not limited to Mg²⁺, washsolutions, and the like).

As illustrated in FIG. 8, the admission of the sequencing reagents tothe flow cell 200 may be accomplished via one or more valves 270 and oneor more pumps 274 that are controlled by computer 260. A number oftechniques may be used to admit (i.e., introduce) the various processingmaterials (i.e., solutions, samples, reaction reagents, wash solutions,and the like) to the wells of such a flow cell. As illustrated in FIG.8, reagents including bases may be admitted to the flow cell (e.g., viathe computer controlled valve 270 and pumps 274) from which they diffuseinto the wells, or reagents may be added to the flow cell by other meanssuch as an ink jet. In yet another example, the flow cell 200 may notcontain any wells, and diffusion properties of the reagents may beexploited to limit cross-talk between respective sensors of the ISFETarray 100.

As will be discussed in greater detail herein, various embodiments ofthe sequencing methods provided in accordance with the invention usebeads having attached to their surface multiple identical copies of atemplate nucleic acid. Such beads are generally referred to herein as“loaded” with nucleic acid. Preferably, each reaction well comprisesonly a single bead. The nucleic acid loaded beads, of which there may betens, hundreds, thousands, or more, first enter the flow cell and thenindividual beads enter individual wells. The beads may enter the wellspassively or otherwise. For example, the beads may enter the wellsthrough gravity without any applied external force. The beads may enterthe wells through an applied external force including but not limited toa magnetic force or a centrifugal force. In some embodiments, if anexternal force is applied, it is applied in a direction that is parallelto the well height/depth rather than transverse to the wellheight/depth, with the aim being to “capture” as many beads as possible.Preferably, the wells (or well arrays) are not agitated, as for examplemay occur through an applied external force that is perpendicular to thewell height/depth. Moreover, once the wells are so loaded, they are notsubjected to any other force that could dislodge the beads from thewells.

Furthermore, as will be discussed herein, in addition to the nucleicacid loaded beads, each well may also comprise a plurality of smallerbeads, referred to herein as “packing beads”. The packing beads may becomposed of any inert material that does not interact or interfere withanalytes, reagents, reaction parameters, and the like, present in thewells. Packing beads may be positioned between the chemFET surface andthe nucleic acid loaded bead, in which case they may be introduced intothe wells before the nucleic acid loaded beads. Alternatively, they maybe positioned all around the nucleic acid loaded beads, in which casethey may be added to the wells before, during and/or after the nucleicacid loaded beads. In still other embodiments, the majority of thepacking beads may be positioned on top of the nucleic acid loaded beads,in which case they may be added to the wells after the nucleic acidloaded beads.

Packing beads may serve one or more various functions. For example, thepacking beads may act to minimize or prevent altogether dislodgement ofnucleic acid loaded beads from wells, particularly if some fraction ofthem are positioned on top of the nucleic acid loaded beads. The packingbeads may act to decrease the diffusion rate of one or more component inthe solution in the well, including but not limited to PPi andunincorporated nucleotides. In some embodiments, the packing beads areparamagnetic beads. Packing beads are commercially available fromsources such as Bangs Laboratories.

Diffusion may also be impacted by including in the reaction chambersviscosity increasing agents. An example of such an agent is a polymerthat is not a nucleic acid (i.e., a non-nucleic acid polymer). Thepolymer may be naturally or non-naturally occurring, and it may be ofany nature provided it does not interfere with nucleotide incorporationand detection thereof except for slowing the diffusion of PPi,unincorporated nucleotides, and/or other reaction byproducts orreagents, towards the reaction chamber bottom. An example of a suitablepolymer is polyethylene glycol (PEG). Other examples include PEA,dextrans, acrylamides, celluloses (e.g. methyl cellulose), and the like.The polymer may be free in solution or it may be immobilized (covalentlyor non-covalently) to one or more sides of the reaction chamber, to thecapture bead, and/or to any packing beads that may be present.Non-covalent attachment may be accomplished via a biotin-avidininteraction.

In still other sequencing embodiments, instead of employing capturebeads, the wells can be coated with one or more nucleic acids, includingfor example a pair of primer nucleic acids, and the template nucleicacid having adaptor nucleotide sequences complementary to the primernucleotide sequence may be introduced into the wells. These and otheragents useful in immobilizing analytes or template nucleic acids may beprovided to the sensor array 100, to individual dies as part of the chippackaging, or to wells immediately before the processing of a sample).Other methods involving solgels may be used to immobilize agents such asnucleic acids near the surface of the ISFET array 100.

As will be discussed in greater detail herein, in some aspects of thesequencing methods contemplated by the invention, the template nucleicacid may be amplified prior to or after placement in the well. Variousmethods exist to amplify nucleic acids. Thus, in one aspect, once atemplate nucleic acid is loaded into a well of the flow cell 200,amplification may be performed in the well, the resulting amplifiedproduct denatured, and sequencing-by-synthesis or then performed.Amplification methods include but are not limited to bridgeamplification, rolling circle amplification, or other strategies usingisothermal or non-isothermal amplification techniques.

In sum, the flow cell 200 in the system of FIG. 8 may be configured in avariety of manners to provide one or more analytes (or one or morereaction solutions) in proximity to the ISFET array 100. For example, atemplate nucleic acid may be directly attached or applied in suitableproximity to one or more pixels of the sensor array 100, or on a supportmaterial (e.g., one or more “beads”) located above the sensor array.Processing reagents (e.g., enzymes such as polymerases) can also beplaced on the sensors directly, or on one or more solid supports inproximity to the sensors. It is to be understood that the device may beused without wells or beads for a number of biosensor applicationsinvolving the detection and/or measurement of at least onesensor-detectable product (e.g., ion concentration change).

In the system 1000 of FIG. 8, according to one embodiment the ISFETsensor array 100 monitors ionic species, and in particular, changes inthe levels/amounts and/or concentration of ionic species. In importantembodiments, the species are those that result from a nucleic acidsynthesis or sequencing reaction. One particularly important ionicspecies is the PPi that is released as a result of nucleotideincorporation. Another important species is the excess nucleotides addedto and remaining in the reaction chamber once the synthesis orsequencing reaction is complete. Such nucleotides are referred to hereinas “unincorporated nucleotides.”

As will be discussed in greater detail herein, various embodiments ofthe present invention may relate to monitoring/measurement techniquesthat involve the static and/or dynamic responses of an ISFET. In oneembodiment relating to detection of nucleotide incorporation during anucleic acid synthesis or sequencing reaction, detection/measurementtechniques particularly rely on the transient or dynamic response of anISFET (ion-step response, or “ion pulse” output), as discussed above inconnection with FIG. 2A, to detect concentration changes of variousionic species relating to a nucleic acid synthesis or sequencingreaction. Although the particular example of a nucleic acid synthesis orsequencing reaction is provided to illustrate the transient or dynamicresponse of an ISFET, it should be appreciated that according to otherembodiments, the transient or dynamic response of an ISFET as discussedbelow may be exploited for monitoring/sensing other types of chemicaland/or biological activity beyond the specific example of a nucleic acidsynthesis or sequencing reaction.

In one exemplary implementation, beyond the step-wise or essentiallyinstantaneous pH changes in the analyte solution contemplated by priorresearch efforts, detection/measurement techniques relying on thedynamic response of an ISFET according to some embodiments of thepresent invention are based at least in part on the differentialdiffusion of various ionic species proximate to the analyte/passivationlayer interface of the ISFET(s) (e.g., at the bottom of a reaction wellover an ISFET). In particular, Applicants have recognized andappreciated that if a given stimulus constituted by a change in ionicstrength proximate to the analyte/passivation layer interface, due tothe appropriate diffusion of respective species of interest, occurs at arate that is significantly faster than the ability of the passivationlayer to adjust its surface charge density in response to the stimulusof the concentration change (e.g., faster than a characteristic responsetime constant τ associated with the passivation layer surface), astep-wise or essentially instantaneous change in ionic strength is notnecessarily required to observe an ion pulse output from the ISFET. Thisprinciple is applicable not only to the example of DNA sequencing, butalso to other types of chemical and chemical reaction sensing, as well.

For purposes of the present disclosure, the pulse response of an ISFETto a stimulus constituted by any significant change in ionic strength inthe analyte solution that occurs on a sufficiently fast time scale isreferred to as an “ion pulse,” wherein the amplitude of the pulse isdescribed by Eq. (17) above, and wherein the width and shape of thepulse (rise and decay of the pulse over time) relate to the diffusionparameters associated with the ion species giving rise to the change inionic strength and the equilibrium reaction kinetics at theanalyte/passivation layer interface (as reflected in one or morecharacteristic time constants τ, as discussed above in connection withEq. (18)). In various embodiments discussed herein, one or more ionpulses due to concentration changes of ionic species proximate to theanalyte/passivation layer surface may be observed in an ISFET outputsignal if such concentration changes occur at a rate that is faster thanthe ability of the passivation layer to adjust its surface chargedensity in response to the stimulus of the concentration change (e.g.,ionic strength changes occur in a time t<<τ). Again, from the foregoing,it should be appreciated that step-wise or instantaneous changes inionic strength (e.g., a high flow rate directed normal to the ISFETpassivation layer surface) is not required to observe an ion pulseoutput from the ISFET.

More specifically, with reference again to Eqs. (10) and (11) (assumingthe applicability of the Debye theory in a regime of relatively lowerionic strengths, e.g., <1 mole/liter), consider that an ISFETre-equilibrates to the same surface potential after a change in ionicstrength. This suggests that if the Debye screening length changes toλ′, the surface charge density changes by the opposite amount to keepthe surface potential constant, i.e.:

$\begin{matrix}{\sigma^{\prime} = {\frac{\sigma\lambda}{\lambda^{\prime}}.}} & (20)\end{matrix}$As discussed above in connection with Eq. (18), the surface chargedensity σ may be described approximately as an exponential function oftime with time constant τ. Accordingly, if there is a time-varying Debyescreening length λ(t) due to a time varying ionic strength I(t) in theanalyte solution (see Eqs. (12) and (13)), the surface charge densityobeys the differential equation:

$\begin{matrix}{\frac{\mathbb{d}\sigma}{\mathbb{d}t} = {- {\frac{\left\lbrack {\sigma - \frac{{\sigma(0)}{\lambda(0)}}{\lambda(t)}} \right\rbrack}{\tau}.}}} & (21)\end{matrix}$From Eq. (21), it may be observed that the surface charge density iscontinuously trying to get back to a value that gives the initialsurface potential as the Debye screening length changes with time. Thesurface potential as a function of time may be expressed in terms of theDebye screening length. from Eqs. (10) and (11), as:

$\begin{matrix}{{\Psi_{0}(t)} = {\frac{{- {\lambda(t)}}{\sigma(t)}}{k\; ɛ_{0}}.}} & (22)\end{matrix}$Introducing a new variable g(t) that includes σ(t) and referring to Eq.(12), Eq. (22) may be rewritten in terms of a time varying ionicstrength I(t) as:

$\begin{matrix}{{{\Psi_{0}(t)} = \frac{g(t)}{\sqrt{I(t)}}},} & (23)\end{matrix}$from which is obtained:

$\begin{matrix}{\frac{\mathbb{d}g}{\mathbb{d}t} = {- {\frac{\left\lbrack {g - \frac{{g(0)}\sqrt{I(t)}}{\sqrt{I(0)}}} \right\rbrack}{\tau}.}}} & (24)\end{matrix}$Eqs. (23) and (24) show that the generation and overall profile of anion pulse in an ISFET output signal depends both on the time constant τassociated with the surface kinetics of the analyte/passivation layerinterface and the ionic strength as a function of time I(t), which isgiven by the concentrations of respective ionic species in the reactionwell as they change over time due to reaction and diffusion. As notedabove, from the foregoing, Applicants have recognized and appreciatedthat as long as I(t) varies at a rate that is faster than the ability ofthe surface charge density to re-equilibrate (as characterized by thetime constant τ), an ion pulse output may be observed, wherein theprofile of the onset of the pulse is determined primarily by I(t) andthe decay profile of the pulse is determined primarily by the timeconstant τ. Again, the foregoing analysis applies generally to virtuallyany ion species present in the analyte solution and, accordingly, thetransient or dynamic response of an ISFET may be exploited to monitor avariety of chemical and/or biological activity.

With respect to the specific example of a nucleic acid synthesis orsequencing reaction, in one embodiment an exemplary ISFET of the array100 may be employed to provide an output signal that includes one ormore ion pulses in response to addition of synthesis or sequencingreagents to a corresponding reaction well associated with the ISFET andcontaining a previously loaded template. With reference to FIGS. 8A and8B, in an exemplary method according to this embodiment, knownnucleotides (i.e., dNTP, where N refers to one of A, C, G, or T) areintroduced into a reaction well with appropriate diffusion parameters(discussed further below) in order to initiate the synthesis orsequencing reaction, and the number of ion pulses and the time intervalbetween successive pulses (if multiple pulses are generated) detected inthe ISFET output signal provide valuable information about nucleotideincorporation.

More specifically, in a method according to one embodiment for detectingnucleotide incorporation, if one or more of the nucleotides introducedinto a reaction well are incorporated upon their introduction, PPi isgenerated. FIG. 8A is a graph showing the concentration (in μM) ofvarious ionic species as a function of time in the analyte proximate theISFET analyte/passivation layer interface during a nucleic acidsynthesis or sequencing reaction in one exemplary implementation. In theparticular example shown in FIG. 8A, the plot 41 indicates PPiconcentration as a function of time, and the plot 43 indicates dNTPconcentration as a function of time (in seconds). With time t=0 secondscorresponding essentially to an initial concentration increase of PPi,and with particular diffusion parameters for the species of interest, itmay be observed that at approximately 15 sec<t<17.5 sec theconcentration of PPi suddenly decreases and there is a correspondingincrease in the concentration of dNTP. For purposes of the presentdiscussion, each incorporation of a given nucleotide type dNTP uponadmission to a reaction well is referred to as an “incorporation event.”In the example of FIG. 8A, as discussed further below, multipleincorporation events occur during the time period 0<t<17.5 in which thePPi concentration is elevated.

FIG. 8B is a graph illustrating multiple plots of ISFET output voltageas a function of time for exemplary nucleic acid sequencing/synthesisreactions similar to those profiled in FIG. 8A. With reference to Table1 above, and Eqs. (3), (4), (5) and (16), it may be appreciated thatbased on a passivation layer of either silicon dioxide or siliconnitride, for example, and an analyte pH of approximately 8-9, a“baseline” ISFET output voltage on the order of approximately −200 mV isto be expected, as illustrated in FIG. 8B. Given appropriate diffusionparameters of the ionic species involved, the initial generation of PPiproximate to the analyte/passivation layer interface of the ISFET due toone or more incorporation events constitutes a sufficiently fast ionicstrength change stimulus I(t), thereby causing a change in the surfacepotential Ψ₀ according to Eq. (23) above and a corresponding change inthe ISFET output signal given by Eqs. (3), (4) and (5) above, resultingin an ion pulse in the ISFET output signal. Stated differently,irrespective of the duration of PPi generation due to one or moreincorporation events, following initial PPi generation constituting asufficiently fast stimulus, the analyte/passivation layer interfacereturns to some steady-state equilibrium, thereby completing thetransient response to the initial stimulus and giving rise to a pulse inthe ISFET signal, examples of which are illustrated in FIG. 8B. It maybe observed generally from FIG. 8B that in one exemplary implementation,the time constant τ for the decay profile a given ion pulse may be onthe order of approximately 2 to 2.5 seconds. However, it should beappreciated that various embodiments of the present invention are notlimited to this illustrative example, as the time constant τ may dependat least in part on material-dependent properties of a given passivationlayer as well as the bulk pH of the analyte solution (e.g., as discussedabove in connection with Eq. (19)).

In FIG. 8B, a number of different plots for an ISFET output signal as afunction of time are indicated, with the legend employing the notationN_(poly)=x (x=0, 1, 2, . . . 7) to indicate the number of incorporationevents giving rise to the indicated output signal. All of the plots ofoutput signals corresponding to one or more incorporation events(N_(poly)≠0) include two pulses whose respective peaks are separated bysome time interval. For example, the plot of the output signal includingthe pulses 45 and 49 separated by the time interval 47 corresponds tothe concentration changes illustrated in FIG. 8A and is labeled with thelegend notation N_(poly)=7, indicating that the time interval 47 in thisexemplary implementation represents seven incorporation events.

More specifically, in an exemplary method to detect nucleotideincorporation, if PPi is generated pursuant to one or more incorporationevents, at some point incorporation ceases, PPi is no longer generated,and the concentration of unincorporated nucleotide (dNTP) increases, asshown in FIG. 8A by the plot 43. If diffusion parameters are such thatthe increased concentration of dNTP occurs after completion of the ionpulse due to PPi (i.e., after the analyte/passivation layer interfacehas reached steady state following the initial PPi stimulus), thisincreased concentration of unincorporated nucleotide constitutes asecond ion stimulus that generates a second ion pulse in the ISFEToutput signal; again, using the illustrative example of concentrationchanges shown in FIG. 8A corresponding to seven incorporation events,the plot N_(p01)=7 in FIG. 8B includes a first ion pulse 45corresponding to initial PPi generation, and a second pulse 49corresponding to the increased concentration of dNTP once incorporationevents cease, whose respective peaks are separated by the time interval47.

More generally, Applicants have recognized and appreciated that for anyof the ISFET output signals in FIG. 8B including multiple pulsesseparated by some time interval, the interval of time between a firstion pulse due to PPi generation and a second ion pulse due to increasedconcentration of unincorporated nucleotide relates at least in part tothe number of incorporation events. As discussed further below, thistime interval also may relate at least in part to other parametersincluding, but not limited to, dimensions of the reaction well, packingdensity within the well, kinetics of the nucleotide sequencing orsynthesis reaction, diffusion coefficients of the involved species,nucleotide concentration, and initial amount of DNA. In addition to theoutput signal associated with the legend N_(poly)=7 in FIG. 8B, forpurposes of further illustration a number of other pulses are labeled inFIG. 8B; in particular, the pulse 53 constitutes the second of twopulses in an output signal labeled as N_(poly)=3 (i.e., the timeinterval between the pulse 45 and the pulse 53 corresponds to threeincorporation events), and the pulse 55 constitutes the second of twopulses in an output signal labeled as N_(poly)=5 (i.e., the timeinterval between the pulse 45 and the pulse 55 corresponds to fiveincorporation events).

In another aspect of the foregoing method for detecting nucleotideincorporation, if no nucleotides are incorporated upon introduction of agiven nucleotide dNTP into a well, no PPi is generated, and the firstand only ion stimulus to the ISFET associated with the well is the risein concentration of dNTP as it is admitted to the well. As a result,only a single ion pulse is generated, again assuming appropriatediffusion parameters for the dNTP. This situation is illustrated by theoutput signal labeled as N_(poly)=0 in FIG. 8B, which includes only asingle pulse 51 in the output signal.

Accordingly, the above-described method provides for significantcharacterization of the nucleic acid synthesis or sequencing reaction asfollows:

-   -   1) If only a single ion pulse is present in the ISFET output        signal, there is no incorporation of the nucleotide introduced        to a reaction well (i.e., only dNTP stimulus);    -   2) If two successive ion pulses are present in the ISFET output        signal, there is incorporation of the nucleotide introduced to        the reaction well (i.e., PPi stimulus followed by dNTP        stimulus); and    -   3) The time interval between two successive ion pulses is a        function of the number of incorporation events.

In various exemplary implementations of methods pursuant to the variousconcepts discussed immediately above for nucleotide incorporationdetection, it should be appreciated that merely determining whether oneor two pulses are present in an ISFET output signal provides usefulinformation regarding nucleotide incorporation. Hence, not all methodsaccording to the present invention need also analyze the time intervalbetween multiple pulses, if present. Again, however, this time intervalprovides additional useful information relating to the number ofincorporation events, and indeed some exemplary implementations analyzenot only the number of pulses present in an ISFET output signal but thetiming between pulses, in some instances taking into consideration thevarious diffusion parameters involved for the respective species givingrise to one or more ion pulses.

Regarding the method for nucleotide incorporation detection discussedabove, and with reference again to Eq. (17) above, Applicants haverecognized and appreciated that various parameters of a nucleic acidsynthesis or sequencing reaction may be improved and/or optimized toincrease the signal-to-noise ratio (SNR) associated with a given ionpulse in an ISFET output signal (e.g., increase the amplitude ΔΨ₀ of theion pulse).

First, it may be appreciated from Eq. (17) that a higher initialequilibrium surface potential Ψ₁, (e.g., the steady state surfacepotential in the presence of a wash buffer before introduction of agiven nucleotide dNTP to a reaction well), generally results in a higheramplitude ΔΨ₀ for an ion pulse. In one aspect, with reference again toEq. (16), Applicants have appreciated that a higher Ψ₁ is facilitated bythe selection of a wash buffer having a pH that is significantlydifferent than the pH_(pzc) of the ISFET's passivation layer (i.e., thebulk analyte pH that results in zero charge density at the passivationlayer surface). Hence, in various implementations, wash buffers may beappropriately selected based on the type of material employed for thepassivation layer.

From Eq. (15), Applicants also have appreciated that a relatively highpH sensitivity (ΔΨ₀/ΔpH) of the ISFET also may contribute to a higherΨ₁; in this manner, as discussed above in connection with Eq. (15), apassivation layer material with a large intrinsic buffering capacityβ_(int) may be desirable, as the dimensionless sensitivity factor αgiven in Eq. (15) is increased by a large β_(int). It should also beappreciated in this regard, however, that a high sensitivity to pH isnot necessarily a requirement in the nucleotide incorporation detectionmethod according to the specific embodiment described above, as themethod is predicated primarily on detection of PPi and dNTPconcentration changes, and not measurement of hydrogen ion concentrationper se. This situation underscores the applicability to the methoddescribed above of various ISFET array optimization techniques discussedherein based on a consideration of a more limited pH measurement rangeand reduced linearity requirements (e.g., in consideration of the MOSFETbody effect). Of course, methods according to other embodiments of theinvention discussed herein indeed may rely on hydrogen ion concentrationand concentration changes as reaction indicators. In any case, as notedabove, Applicants have appreciated that a relatively high pH sensitivityof the ISFET, even over a significantly limited pH range, may in someinstances facilitate an increased amplitude ΔΨ₀ for an ion pulse andhence an increased SNR of the ISFET output signal.

Additionally, Applicants have appreciated from Eq. (17) that reducing orminimizing the initial double layer capacitance C_(dl,1) due to theinitial ionic strength in the analyte solution (i.e., arising from thewash buffer, prior to introduction of dNTP), and/or increasing ormaximizing the subsequent double layer capacitance C_(dl,2) arising fromthe concentration change stimulus (i.e., PPi generation or dNTPincrease), also results in a higher amplitude ΔΨ₀ for an ion pulse.Accordingly, in various implementations, low ionic strength buffers maybe appropriately selected to facilitate an increased amplitude ΔΨ₀ foran ion pulse and hence an increased SNR of the ISFET output signal.

With respect to time resolution, as alluded to above, some aspect ofpulse shape as well as the time interval between any two pulses is afunction of the time and rate at which various ionic species of interestare generated proximate to the ISFET's analyte/passivation layerinterface. Examples of significant parameters in these respects include,but are not limited to, the flow rate at which various constituents ofthe analyte solution (e.g., various reagents) are admitted to thereaction well, respective and/or collective concentration of variousspecies, amount of template material present in the well, kinetics ofthe nucleic acid sequencing or synthesis reactions that may occur in thewell, and various factors that affect diffusion of species in thereaction well (e.g., diffusion coefficients of various species,geometry/dimensions of the reaction well, presence and size of packingbeads, additives to the analyte solution that affect density).Parameters generally relating to time resolution of one or more ionpulses in an ISFET output signal are discussed in greater detail below.

As noted above, the ISFET may be employed to measure steady state pHvalues, since in some embodiments pH change is proportional to thenumber of nucleotides incorporated into the newly synthesized nucleicacid strand. In other embodiments discussed in greater detail below, theFET sensor array may be particularly configured for sensitivity to otheranalytes that may provide relevant information about the chemicalreactions of interest. An example of such a modification orconfiguration is the use of analyte-specific receptors to bind theanalytes of interest, as discussed in greater detail herein.

Via an array controller 250 (also under operation of the computer 260),the ISFET array may be controlled so as to acquire data (e.g., outputsignals of respective ISFETs of the array) relating to analyte detectionand/or measurements, and collected data may be processed by the computer260 to yield meaningful information associated with the processing(including sequencing) of the template nucleic acid.

With respect to the ISFET array 100 of the system 1000 shown in FIG. 8,in one embodiment the array 100 is implemented as an integrated circuitdesigned and fabricated using standard CMOS processes (e.g., 0.35micrometer process, 0.18 micrometer process), comprising all the sensorsand electronics needed to monitor/measure one or more analytes and/orreactions. With reference again to FIG. 1, one or more referenceelectrodes 76 to be employed in connection with the ISFET array 100 maybe placed in the flow cell 200 (e.g., disposed in “unused” wells of theflow cell) or otherwise exposed to a reference (e.g., one or more of thesequencing reagents 172) to establish a base line against which changesin analyte concentration proximate to respective ISFETs of the array 100are compared. The reference electrode(s) 76 may be electrically coupledto the array 100, the array controller 250 or directly to the computer260 to facilitate analyte measurements based on voltage signals obtainedfrom the array 100; in some implementations, the reference electrode(s)may be coupled to an electric ground or other predetermined potential,or the reference electrode voltage may be measured with respect toground, to establish an electric reference for ISFET output signalmeasurements, as discussed further below.

The ISFET array 100 is not limited to any particular size, as one- ortwo-dimensional arrays, including but not limited to as few as two to256 pixels (e.g., 16 by 16 pixels in a two-dimensional implementation)or as many as 54 mega-pixels (e.g., 7400 by 7400 pixels in atwo-dimensional implementation) or even greater may be fabricated andemployed for various chemical/biological analysis purposes pursuant tothe concepts disclosed herein. In one embodiment of the exemplary systemshown in FIG. 8, the individual ISFET sensors of the array may beconfigured for sensitivity to PPi, unincorporated nucleotides, hydrogenions, and the like; however, it should also be appreciated that thepresent disclosure is not limited in this respect, as individual sensorsof an ISFET sensor array may be particularly configured for sensitivityto other types of ion concentrations for a variety of applications(materials sensitive to other ions such as sodium, silver, iron,bromine, iodine, calcium, and nitrate, for example, are known).

More generally, a chemFET array according to various embodiments of thepresent disclosure may be configured for sensitivity to any one or moreof a variety of analytes. In one embodiment, one or more chemFETs of anarray may be particularly configured for sensitivity to one or moreanalytes and/or one or more binding events, and in other embodimentsdifferent chemFETs of a given array may be configured for sensitivity todifferent analytes. For example, in one embodiment, one or more sensors(pixels) of the array may include a first type of chemFET configured tobe sensitive to a first analyte, and one or more other sensors of thearray may include a second type of chemFET configured to be sensitive toa second analyte different from the first analyte. In one exemplaryimplementation, both a first and a second analyte may indicate aparticular reaction such as for example nucleotide incorporation in asequencing-by-synthesis method. Of course, it should be appreciated thatmore than two different types of chemFETs may be employed in any givenarray to detect and/or measure different types of analytes and/or otherreactions. In general, it should be appreciated in any of theembodiments of sensor arrays discussed herein that a given sensor arraymay be “homogeneous” and include chemFETs of substantially similar oridentical types to detect and/or measure a same type of analyte (e.g.,pH or other ion concentration), or a sensor array may be “heterogeneous”and include chemFETs of different types to detect and/or measuredifferent analytes. For simplicity of discussion, again the example ofan ISFET is discussed below in various embodiments of sensor arrays, butthe present disclosure is not limited in this respect, and several otheroptions for analyte sensitivity are discussed in further detail below(e.g., in connection with FIG. 11A).

The chemFET arrays configured for sensitivity to any one or more of avariety of analytes may be disposed in electronic chips, and each chipmay be configured to perform one or more different biological reactions.The electronic chips can be connected to the portions of theabove-described system which read the array output by means of pinscoded in a manner such that the pins convey information to the system asto characteristics of the array and/or what kind of biologicalreaction(s) is(are) to be performed on the particular chip.

In one embodiment, the invention encompasses an electronic chipconfigured for conducting biological reactions thereon, comprising oneor more pins for delivering information to a circuitry identifying acharacteristic of the chip and/or a type of reaction to be performed onthe chip. Such t may include, but are not limited to, a short nucleotidepolymorphism detection, short tandem repeat detection, or sequencing.

In another embodiment, the invention encompasses a system adapted toperforming more than one biological reaction on a chip comprising: achip receiving module adapted for receiving the chip; and a receiver fordetecting information from the electronic chip, wherein the informationdetermines a biological reaction to be performed on the chip. Typically,the system further comprises one or more reagents to perform theselected biological reaction.

In another embodiment, the invention encompasses an apparatus forsequencing a polymer template comprising: at least one integratedcircuit that is configured to relay information about spatial locationof a reaction chamber, type of monomer added to the spatial location,time required to complete reaction of a reagent comprising a pluralityof the monomers with an elongating polymer.

In exemplary implementations based on 0.35 micrometer CMOS processingtechniques (or CMOS processing techniques capable of smaller featuresizes), each pixel of the ISFET array 100 may include an ISFET andaccompanying enable/select components, and may occupy an area on asurface of the array of approximately ten micrometers by ten micrometers(i.e., 100 micrometers²) or less; stated differently, arrays having apitch (center of pixel-to-center of pixel spacing) on the order of 10micrometers or less may be realized. An array pitch on the order of 10micrometers or less using a 0.35 micrometer CMOS processing techniqueconstitutes a significant improvement in terms of size reduction withrespect to prior attempts to fabricate ISFET arrays, which resulted inpixel sizes on the order of at least 12 micrometers or greater.

More specifically, in some embodiments discussed further below based onthe inventive concepts disclosed herein, an array pitch of approximatelynine (9) micrometers allows an ISFET array including over 256,000 pixels(e.g., a 512 by 512 array), together with associated row and columnselect and bias/readout electronics, to be fabricated on a 7 millimeterby 7 millimeter semiconductor die, and a similar sensor array includingover four million pixels (e.g., a 2048 by 2048 array yielding over 4Mega-pixels) to be fabricated on a 21 millimeter by 21 millimeter die.In other examples, an array pitch of approximately 5 micrometers allowsan ISFET array including approximately 1.55 Mega-pixels (e.g., a 1348 by1152 array) and associated electronics to be fabricated on a 9millimeter by 9 millimeter die, and an ISFET sensor array including over14 Mega-pixels and associated electronics on a 22 millimeter by 20millimeter die. In yet other implementations, using a CMOS fabricationprocess in which feature sizes of less than 0.35 micrometers arepossible (e.g., 0.18 micrometer CMOS processing techniques), ISFETsensor arrays with a pitch significantly below 5 micrometers may befabricated (e.g., array pitch of 2.6 micrometers or pixel area of lessthan 8 or 9 micrometers²), providing for significantly dense ISFETarrays. Of course, it should be appreciated that pixel sizes greaterthan 10 micrometers (e.g., on the order of approximately 20, 50, 100micrometers or greater) may be implemented in various embodiments ofchemFET arrays according to the present disclosure also.

As will be understood by those of skill in the art, the ability tominiaturize sequencing reactions reduces the time, cost and laborinvolved in sequencing of large genomes (such as the human genome).

In other aspects of the system shown in FIG. 8, one or more arraycontrollers 250 may be employed to operate the ISFET array 100 (e.g.,selecting/enabling respective pixels of the array to obtain outputsignals representing analyte measurements). In various implementations,one or more components constituting one or more array controllers may beimplemented together with pixel elements of the arrays themselves, onthe same integrated circuit (IC) chip as the array but in a differentportion of the IC chip, or off-chip. In connection with array control,analog-to-digital conversion of ISFET output signals may be performed bycircuitry implemented on the same integrated circuit chip as the ISFETarray, but located outside of the sensor array region (locating theanalog to digital conversion circuitry outside of the sensor arrayregion allows for smaller pitch and hence a larger number of sensors, aswell as reduced noise). In various exemplary implementations discussedfurther below, analog-to-digital conversion can be 4-bit, 8-bit, 12-bit,16-bit or other bit resolutions depending on the signal dynamic rangerequired.

As used herein, an array is planar arrangement of elements such assensors or wells. The array may be one or two dimensional. A onedimensional array is an array having one column (or row) of elements inthe first dimension and a plurality of columns (or rows) in the seconddimension. An example of a one dimensional array is a 1×5 array. A twodimensional array is an array having a plurality of columns (or rows) inboth the first and the second dimension. The number of columns (or rows)in the first and second dimensions may or may not be the same. Anexample of a two dimensional array is a 5×10 array.

Having provided a general overview of the role of a chemFET (e.g.,ISFET) array 100 in an exemplary system 1000 for measuring one or moreanalytes, following below are more detailed descriptions of exemplarychemFET arrays according to various inventive embodiments of the presentdisclosure that may be employed in a variety of applications. Again, forpurposes of illustration, chemFET arrays according to the presentdisclosure are discussed below using the particular example of an ISFETarray, but other types of chemFETs may be employed in alternativeembodiments. Also, again, for purposes of illustration, chemFET arraysare discussed in the context of nucleic acid sequencing applications,however, the invention is not so limited and rather contemplates avariety of applications for the chemFET arrays described herein.

As noted above, various inventive embodiments disclosed hereinspecifically improve upon the ISFET array design of Milgrew et al.discussed above in connection with FIGS. 1-7, as well as other priorISFET array designs, so as to significantly reduce pixel size and arraypitch, and thereby increase the number of pixels of an ISFET array for agiven semiconductor die size (i.e., increase pixel density). In someimplementations, an increase in pixel density is accomplished while atthe same time increasing the signal-to-noise ratio (SNR) of outputsignals corresponding to respective measurements relating to one or moreanalytes and the speed with which such output signals may be read fromthe array. In particular, Applicants have recognized and appreciatedthat by relaxing requirements for ISFET linearity and focusing on a morelimited signal output/measurement range (e.g., signal outputscorresponding to a pH range of from approximately 7 to 9 or smallerrather than 1 to 14, as well as output signals that may not necessarilyrelate significantly to pH changes in sample), individual pixelcomplexity and size may be significantly reduced, thereby facilitatingthe realization of very large scale dense ISFET arrays.

To this end, FIG. 9 illustrates one column 102 _(j) of an ISFET array100, according to one inventive embodiment of the present disclosure, inwhich ISFET pixel design is appreciably simplified to facilitate smallpixel size. The column 102 _(j) includes n pixels, the first and last ofwhich are shown in FIG. 9 as the pixels 105 ₁ and 105 _(n). As discussedfurther below in connection with FIG. 13, a complete two-dimensionalISFET array 100 based on the column design shown in FIG. 9 includes msuch columns 102 _(j) (j=1, 2, 3, . . . m) with successive columns ofpixels generally arranged side by side. Of course, the ISFETs may bearrayed in other than a row-column grid, such as in a honeycomb pattern.

In one aspect of the embodiment shown in FIG. 9, each pixel 105 ₁through 105 _(n) of the column 102 _(j) includes only three components,namely, an ISFET 150 (also labeled as Q1) and two MOSFET switches Q2 andQ3. The MOSFET switches Q2 and Q3 are both responsive to one of n rowselect signals ( RowSel₁ through RowSel_(n) , logic low active) so as toenable or select a given pixel of the column 102 _(j). Using pixel 105 ₁as an example that applies to all pixels of the column, the transistorswitch Q3 couples a controllable current source 106 _(j) via the line112 ₁ to the source of the ISFET 150 upon receipt of the correspondingrow select signal via the line 118 ₁. The transistor switch Q2 couplesthe source of the ISFET 150 to column bias/readout circuitry 110 _(j)via the line 114 ₁ upon receipt of the corresponding row select signal.The drain of the ISFET 150 is directly coupled via the line 116 ₁ to thebias/readout circuitry 110 _(j). Thus, only four signal lines per pixel,namely the lines 112 ₁, 114 ₁, 116 ₁ and 118 ₁, are required to operatethe three components of the pixel 105 ₁. In an array of m columns, agiven row select signal is applied simultaneously to one pixel of eachcolumn (e.g., at same positions in respective columns).

As illustrated in FIG. 9, the design for the column 102 _(j) accordingto one embodiment is based on general principles similar to thosediscussed above in connection with the column design of Milgrew et al.shown FIG. 3. In particular, the ISFET of each pixel, when enabled, isconfigured with a constant drain current I_(Dj) and a constantdrain-to-source voltage V_(DSj) to obtain an output signal V_(Sj) froman enabled pixel according to Eq. (3) above. To this end, the column 102_(j) includes a controllable current source 106 _(j), coupled to ananalog circuitry positive supply voltage VDDA and responsive to a biasvoltage VB1, that is shared by all pixels of the column to provide aconstant drain current I_(Dj) to the ISFET of an enabled pixel. In oneaspect, the current source 106 _(j) is implemented as a current mirrorincluding two long-channel length and high output impedance MOSFETs. Thecolumn also includes bias/readout circuitry 110 _(j) that is also sharedby all pixels of the column to provide a constant drain-to-sourcevoltage to the ISFET of an enabled pixel. The bias/readout circuitry 110is based on a Kelvin Bridge configuration and includes two operationalamplifiers 107A (A1) and 107B (A2) configured as buffer amplifiers andcoupled to analog circuitry positive supply voltage VDDA and the analogsupply voltage ground VSSA. The bias/readout circuitry also includes acontrollable current sink 108 _(j) (similar to the current source 106 j)coupled to the analog ground VSSA and responsive to a bias voltage VB2,and a diode-connected MOSFET Q6. The bias voltages VB1 and VB2 areset/controlled in tandem to provide a complimentary source and sinkcurrent. The voltage developed across the diode-connected MOSFET Q6 as aresult of the current drawn by the current sink 108 _(j) is forced bythe operational amplifiers to appear across the drain and source of theISFET of an enabled pixel as a constant drain-source voltage V_(DSj).

By employing the diode-connected MOSFET Q6 in the bias/readout circuitry110 of FIG. 9, rather than the resistor R_(SDj) as shown in the designof Milgrew et al. illustrated in FIG. 3, a significant advantage isprovided in a CMOS fabrication process; specifically, matching resistorscan be fabricated with error tolerances generally on the order of ±20%,whereas MOSFET matching in a CMOS fabrication process is on the order of±1% or better. The degree to which the component responsible forproviding a constant ISFET drain-to-source voltage V_(DSj) can bematched from column to column significantly affects measurement accuracy(e.g., offset) from column to column. Thus, employing the MOSFET Q6rather than a resistor appreciably mitigates measurement offsets fromcolumn-to-column. Furthermore, whereas the thermal drift characteristicsof a resistor and an ISFET may be appreciably different, the thermaldrift characteristics of a MOSFET and ISFET are substantially similar,if not virtually identical; hence, any thermal drift in MOSFET Q6virtually cancels any thermal drift from ISFET Q1, resulting in greatermeasurement stability with changes in array temperature.

In FIG. 9, the column bias/readout circuitry 110 j also includessample/hold and buffer circuitry to provide an output signal V_(COLj)from the column. In particular, after one of the pixels 105 ₁ through105 _(n) is enabled or selected via the transistors Q2 and Q3 in eachpixel, the output of the amplifier 107A (A1), i.e., a buffered V_(Sj),is stored on a column sample and hold capacitor C_(sh) via operation ofa switch (e.g., a transmission gate) responsive to a column sample andhold signal COL SH. Examples of suitable capacitances for the sample andhold capacitor include, but are not limited to, a range of fromapproximately 500 fF to 2 pF. The sampled voltage is buffered via acolumn output buffer amplifier 111 j (BUF) and provided as the columnoutput signal V_(COLj). As also shown in FIG. 9, a reference voltageVREF may be applied to the buffer amplifier 111 j, via a switchresponsive to a control signal CAL, to facilitate characterization ofcolumn-to-column non-uniformities due to the buffer amplifier 111 j andthus allow post-read data correction.

FIG. 9A illustrates an exemplary circuit diagram for one of theamplifiers 107A of the bias/readout circuitry 110 j (the amplifier 107Bis implemented identically), and FIG. 9B is a graph of amplifier biasvs. bandwidth for the amplifiers 107A and 107B. As shown in FIG. 9A, theamplifier 107A employs an arrangement of multiple current mirrors basedon nine MOSFETs (M1 through M9) and is configured as a unity gainbuffer, in which the amplifier's inputs and outputs are labeled forgenerality as IN+ and VOUT, respectively. The bias voltage VB4(representing a corresponding bias current) controls the transimpedanceof the amplifier and serves as a bandwidth control (i.e., increasedbandwidth with increased current). With reference again to FIG. 9, dueto the sample and hold capacitor C_(sh), the output of the amplifier107A essentially drives a filter when the sample and hold switch isclosed. Accordingly, to achieve appreciably high data rates, the biasvoltage VB4 may be adjusted to provide higher bias currents andincreased amplifier bandwidth. From FIG. 9B, it may be observed that insome exemplary implementations, amplifier bandwidths of at least 40 MHzand significantly greater may be realized. In some implementations,amplifier bandwidths as high as 100 MHz may be appropriate to facilitatehigh data acquisition rates and relatively lower pixel sample or “dwell”times (e.g., on the order of 10 to 20 microseconds).

In another aspect of the embodiment shown in FIG. 9, unlike the pixeldesign of Milgrew et al. shown in FIG. 3, the pixels 105 ₁ through 105_(n) do not include any transmission gates or other devices that requireboth n-channel and p-channel FET components; in particular, the pixels105 ₁ through 105 _(n) of this embodiment include only FET devices of asame type (i.e., only n-channel or only p-channel). For purposes ofillustration, the pixels 105 ₁ and 105 _(n) illustrated in FIG. 9 areshown as comprising only p-channel components, i.e., two p-channelMOSFETs Q2 and Q3 and a p-channel ISFET 150. By not employing atransmission gate to couple the source of the ISFET to the bias/readoutcircuitry 110 _(j), some dynamic range for the ISFET output signal(i.e., the ISFET source voltage V_(S)) may be sacrificed. However,Applicants have recognized and appreciated that by potentially foregoingsome output signal dynamic range (and thereby potentially limitingmeasurement range for a given static and/or dynamic chemical property,such as pH or concentration changes of other ion species), therequirement of different type FET devices (both n-channel and p-channel)in each pixel may be eliminated and the pixel component count reduced.As discussed further below in connection with FIGS. 10-12, thissignificantly facilitates pixel size reduction. Thus, in one aspect,there is a beneficial tradeoff between reduced dynamic range and smallerpixel size.

In yet another aspect of the embodiment shown in FIG. 9, unlike thepixel design of Milgrew et al., the ISFET 150 of each pixel 105 ₁through 105 _(n) does not have its body connection tied to its source(i.e., there is no electrical conductor coupling the body connection andsource of the ISFET such that they are forced to be at the same electricpotential during operation). Rather, the body connections of all ISFETsof the array are tied to each other and to a body bias voltage V_(BODY).While not shown explicitly in FIG. 9, the body connections for theMOSFETs Q2 and Q3 likewise are not tied to their respective sources, butrather to the body bias voltage V_(BODY). In one exemplaryimplementation based on pixels having all p-channel components, the bodybias voltage V_(BODY) is coupled to the highest voltage potentialavailable to the array (e.g., VDDA), as discussed further below inconnection with FIG. 17.

By not tying the body connection of each ISFET to its source, thepossibility of some non-zero source-to-body voltage V_(SB) may give riseto the “body effect,” as discussed above in connection with FIG. 1,which affects the threshold voltage V_(TH) of the ISFET according to anonlinear relationship (and thus, according to Eqs. (3), (4) and (5) mayaffect detection and/or measurement of analyte activity giving rise tosurface potential changes at the analyte/passivation layer interface).However, Applicants have recognized and appreciated that by focusing ona reduced ISFET output signal dynamic range, any body effect that mayarise in the ISFET from a non-zero source-to-body voltage may berelatively minimal. Thus, any measurement nonlinearity that may resultover the reduced dynamic range may be ignored as insignificant or takeninto consideration and compensated (e.g., via array calibration and dataprocessing techniques, as discussed further below in connection withFIG. 17). Applicants have also recognized and appreciated that by nottying each ISFET source to its body connection, all of the FETsconstituting the pixel may share a common body connection, therebyfurther facilitating pixel size reduction, as discussed further below inconnection with FIGS. 10-12. Accordingly, in another aspect, there is abeneficial tradeoff between reduced linearity and smaller pixel size.

FIG. 10 illustrates a top view of a chip layout design for the pixel 105₁ shown in FIG. 9, according to one inventive embodiment of the presentdisclosure. FIG. 11A shows a composite cross-sectional view along theline I-I of the pixel shown in FIG. 10, including additional elements onthe right half of FIG. 10 between the lines II-II and III-III,illustrating a layer-by-layer view of the pixel fabrication, and FIGS.12A through 12L provide top views of each of the fabrication layersshown in FIG. 11A (the respective images of FIGS. 12A through 12L aresuperimposed one on top of another to create the pixel chip layoutdesign shown in FIG. 10). In one exemplary implementation, the pixeldesign illustrated in FIGS. 10-12 may be realized using a standard4-metal, 2-poly, 0.35 micrometer CMOS process to provide a geometricallysquare pixel having a dimension “e” as shown in FIG. 10 of approximately9 micrometers, and a dimension “f” corresponding to the ISFET sensitivearea of approximately 7 micrometers.

In the top view of FIG. 10, the ISFET 150 (labeled as Q1 in FIG. 10)generally occupies the right center portion of the pixel illustration,and the respective locations of the gate, source and drain of the ISFETare indicated as Q1 _(G), Q1 _(S) and Q1 _(D). The MOSFETs Q2 and Q3generally occupy the left center portion of the pixel illustration; thegate and source of the MOSFET Q2 are indicated as Q2 _(G) and Q2 _(S),and the gate and source of the MOSFET Q3 are indicated as Q3 _(G) and Q3_(S). In one aspect of the layout shown in FIG. 10, the MOSFETs Q2 andQ3 share a drain, indicated as Q2/3 _(D). In another aspect, it may beobserved generally from the top view of FIG. 10 that the ISFET is formedsuch that its channel lies along a first axis of the pixel (e.g.,parallel to the line I-I), while the MOSFETs Q2 and Q3 are formed suchthat their channels lie along a second axis perpendicular to the firstaxis. FIG. 10 also shows the four lines required to operate the pixel,namely, the line 112 ₁ coupled to the source of Q3, the line 114 ₁coupled to the source of Q2, the line 116 ₁ coupled to the drain of theISFET, and the row select line 118 ₁ coupled to the gates of Q2 and Q3.With reference to FIG. 9, it may be appreciated that all pixels in agiven column share the lines 112, 114 and 116 (e.g., running verticallyacross the pixel in FIG. 10), and that all pixels in a given row sharethe line 118 (e.g., running horizontally across the pixel in FIG. 10);thus, based on the pixel design of FIG. 9 and the layout shown in FIG.10, only four metal lines need to traverse each pixel.

With reference now to the cross-sectional view of FIG. 11A, highly dopedp-type regions 156 and 158 (lying along the line I-I in FIG. 10) inn-well 154 constitute the source (S) and drain (D) of the ISFET, betweenwhich lies a region 160 of the n-well in which the ISFETs p-channel isformed below the ISFETs polysilicon gate 164 and a gate oxide 165.According to one aspect of the inventive embodiment shown in FIGS. 10and 11, all of the FET components of the pixel 105 ₁ are fabricated asp-channel FETs in the single n-type well 154 formed in a p-typesemiconductor substrate 152. This is possible because, unlike the designof Milgrew et al., 1) there is no requirement for a transmission gate inthe pixel; and 2) the ISFETs source is not tied to the n-well's bodyconnection. More specifically, highly doped n-type regions 162 provide abody connection (B) to the n-well 154 and, as shown in FIG. 10, the bodyconnection B is coupled to a metal conductor 322 around the perimeter ofthe pixel 105 ₁. However, the body connection is not directlyelectrically coupled to the source region 156 of the ISFET (i.e., thereis no electrical conductor coupling the body connection and source suchthat they are forced to be at the same electric potential duringoperation), nor is the body connection directly electrically coupled tothe gate, source or drain of any component in the pixel. Thus, the otherp-channel FET components of the pixel, namely Q2 and Q3, may befabricated in the same n-well 154.

In the composite cross-sectional view of FIG. 11A, a highly doped p-typeregion 159 is also visible (lying along the line I-I in FIG. 10),corresponding to the shared drain (D) of the MOSFETs Q2 and Q3. Forpurposes of illustration, a polysilicon gate 166 of the MOSFET Q3 alsois visible in FIG. 11A, although this gate does not lie along the lineI-I in FIG. 10, but rather “behind the plane” of the cross-section alongthe line I-I. However, for simplicity, the respective sources of theMOSFETs Q2 and Q3 shown in FIG. 10, as well as the gate of Q2, are notvisible in FIG. 11A, as they lie along the same axis (i.e.,perpendicular to the plane of the figure) as the shared drain (if shownin FIG. 11A, these elements would unduly complicate the compositecross-sectional view of FIG. 11A).

Above the substrate, gate oxide, and polysilicon layers shown in FIG.11A, a number of additional layers are provided to establish electricalconnections to the various pixel components, including alternating metallayers and oxide layers through which conductive vias are formed.Pursuant to the example of a 4-Metal CMOS process, these layers arelabeled in FIG. 11A as “Contact,” “Metal1,” “Via1,” “Metal2,” “Via2,”“Metal3,” “Via3,” and “Metal4.” (Note that more or fewer metal layersmay be employed.) To facilitate an understanding particularly of theISFET electrical connections, the composite cross-sectional view of FIG.11A shows additional elements of the pixel fabrication on the right sideof the top view of FIG. 10 between the lines II-II and III-III. Withrespect to the ISFET electrical connections, the topmost metal layer 304corresponds to the ISFETs sensitive area 178, above which is disposed ananalyte-sensitive passivation layer 172. The topmost metal layer 304,together with the ISFET polysilicon gate 164 and the interveningconductors 306, 308, 312, 316, 320, 326 and 338, form the ISFETs“floating gate” structure 170, in a manner similar to that discussedabove in connection with a conventional ISFET design shown in FIG. 1. Anelectrical connection to the ISFETs drain is provided by the conductors340, 328, 318, 314 and 310 coupled to the line 116 ₁. The ISFETs sourceis coupled to the shared drain of the MOSFETs Q2 and Q3 via theconductors 334 and 336 and the conductor 324 (which lies along the lineI-I in FIG. 10). The body connections 162 to the n-well 154 areelectrically coupled to a metal conductor 322 around the perimeter ofthe pixel on the “Metal1” layer via the conductors 330 and 332.

As indicated above, FIGS. 12A through 12L provide top views of each ofthe fabrication layers shown in FIG. 11A (the respective images of FIGS.12A through 12L are superimposed one on top of another to create thepixel chip layout design shown in FIG. 10). In FIG. 12, thecorrespondence between the lettered top views of respective layers andthe cross-sectional view of FIG. 11A is as follows: A) n-type well 154;B) Implant; C) Diffusion; D) polysilicon gates 164 (ISFET) and 166(MOSFETs Q2 and Q3); E) contacts; F) Metal1; G) Via1; H) Metal2; I)Via2; J) Metal3; K) Via3; L) Metal4 (top electrode contacting ISFETgate). The various reference numerals indicated in FIGS. 12A through 12Lcorrespond to the identical features that are present in the compositecross-sectional view of FIG. 11A.

Applicants have recognized and appreciated that, at least in someapplications, pixel capacitance may be a salient parameter for some typeof analyte measurements. Accordingly, in another embodiment related topixel layout and design, various via and metal layers may bereconfigured so as to at least partially mitigate the potential forparasitic capacitances to arise during pixel operation. For example, inone such embodiment, pixels are designed such that there is a greatervertical distance between the signal lines 112 ₁, 114 ₁, 116 ₁ and 118₁, and the topmost metal layer 304 constituting the floating gatestructure 170.

In the embodiment described immediately above, with reference again toFIG. 11A, it may be readily observed that the topmost metal layer 304 isformed in the Metal4 layer (also see FIG. 12L), and the signal lines 112₁, 114 ₁, and 116 ₁ are formed in the Metal3 layer (also see FIG. 12J).Also, while not visible in the view of FIG. 11A, it may be observed fromFIG. 12H that the signal line 118 ₁ is formed in the Metal2 layer. Asone or more of these signals may be grounded from time to time duringarray operation, a parasitic capacitance may arise between any one ormore of these signal lines and metal layer 304. By increasing a distancebetween these signal lines and the metal layer 304, such parasiticcapacitance may be reduced.

To this end, in another embodiment some via and metal layers arereconfigured such that the signal lines 112 ₁, 114 ₁, 116 ₁ and 118 ₁are implemented in the Metal1 and Metal2 layers, and the Metal3 layer isused only as a jumper between the Metal2 layer component of the floatinggate structure 170 and the topmost metal layer 304, thereby ensuring agreater distance between the signal lines and the metal layer 304. FIG.10-1 illustrates a top view of a such a chip layout design for a clusterof four neighboring pixels of an chemFET array shown in FIG. 9, with oneparticular pixel 105 ₁ identified and labeled. FIG. 11A-1 shows acomposite cross-sectional view of neighboring pixels, along the line I-Iof the pixel 105 ₁ shown in FIG. 10-1, including additional elementsbetween the lines II-II, illustrating a layer-by-layer view of the pixelfabrication, and FIGS. 12-1A through 12-1L provide top views of each ofthe fabrication layers shown in FIG. 11A-1 (the respective images ofFIGS. 12-1A through 12-1L are superimposed one on top of another tocreate the pixel chip layout design shown in FIG. 10-1).

In FIG. 10-1, it may be observed that the pixel top view layout isgenerally similar to that shown in FIG. 10. For example, in the topview, the ISFET 150 generally occupies the right center portion of eachpixel, and the MOSFETs Q2 and Q3 generally occupy the left centerportion of the pixel illustration. Many of the component labels includedin FIG. 10 are omitted from FIG. 10-1 for clarity, although the ISFETpolysilicon gate 164 is indicated in the pixel 105 ₁ for orientation.FIG. 10-1 also shows the four lines (112 ₁, 114 ₁, 116 ₁ and 118 ₁)required to operate the pixel. One noteworthy difference between FIG. 10and FIG. 10-1 relates to the metal conductor 322 (located on the Metal1layer) which provides an electrical connection to the body region 162;namely, in FIG. 10, the conductor 322 surrounds a perimeter of thepixel, whereas in FIG. 10-1, the conductor 322 does not completelysurround a perimeter of the pixel but includes discontinuities 727.These discontinuities 727 permit the line 118 ₁ to also be fabricated onthe Metal1 layer and traverse the pixel to connect to neighboring pixelsof a row.

With reference now to the cross-sectional view of FIG. 11A-1, threeadjacent pixels are shown in cross-section, with the center pixelcorresponding to the pixel 105 ₁ in FIG. 10-1 for purposes ofdiscussion. As in the embodiment of FIG. 11A, all of the FET componentsof the pixel 105 ₁ are fabricated as p-channel FETs in the single n-typewell 154. Additionally, as in FIG. 11A, in the composite cross-sectionalview of FIG. 11A-1 the highly doped p-type region 159 is also visible(lying along the line I-I in FIG. 10-1), corresponding to the shareddrain (D) of the MOSFETs Q2 and Q3. For purposes of illustration, thepolysilicon gate 166 of the MOSFET Q3 also is visible in FIG. 11A-1,although this gate does not lie along the line I-I in FIG. 10-1, butrather “behind the plane” of the cross-section along the line I-I.However, for simplicity, the respective sources of the MOSFETs Q2 and Q3shown in FIG. 10-1, as well as the gate of Q2, are not visible in FIG.11A-1, as they lie along the same axis (i.e., perpendicular to the planeof the figure) as the shared drain. Furthermore, to facilitate anunderstanding of the ISFET floating gate electrical connections, thecomposite cross-sectional view of FIG. 11A-1 shows additional elementsof the pixel fabrication between the lines II-II of FIG. 10-1.

More specifically, as in the embodiment of FIG. 11A, the topmost metallayer 304 corresponds to the ISFETs sensitive area 178, above which isdisposed an analyte-sensitive passivation layer 172. The topmost metallayer 304, together with the ISFET polysilicon gate 164 and theintervening conductors 306, 308, 312, 316, 320, 326 and 338, form theISFETs floating gate structure 170. However, unlike the embodiment ofFIG. 11A, an electrical connection to the ISFETs drain is provided bythe conductors 340, 328, and 318, coupled to the line 116 ₁ which isformed in the Metal2 layer rather than the Metal3 layer. Additionally,the lines 112 ₁ and 114 ₁ also are shown in FIG. 11A-1 as formed in theMetal2 layer rather than the Metal3 layer. The configuration of theselines, as well as the line 118 ₁, may be further appreciated from therespective images of FIGS. 12-1A through 12-1L (in which thecorrespondence between the lettered top views of respective layers andthe cross-sectional view of FIG. 11A-1 is the same as that described inconnection with FIGS. 12A-12L); in particular, it may be observed inFIG. 12-1F that the line 118 ₁, together with the metal conductor 322,is formed in the Metal1 layer, and it may be observed that the lines 112₁, 114 ₁ and 116 ₁ are formed in the Metal2 layer, leaving only thejumper 308 of the floating gate structure 170 in the Metal3 layer shownin FIG. 12-1J.

Accordingly, by consolidating the signal lines 112 ₁, 114 ₁, 116 ₁ and118 ₁ to the Metal1 and Metal2 layers and thereby increasing thedistance between these signal lines and the topmost layer 304 of thefloating gate structure 170 in the Metal4 layer, parasitic capacitancesin the ISFET may be at least partially mitigated. It should beappreciated that this general concept (e.g., including one or moreintervening metal layers between signal lines and topmost layer of thefloating gate structure) may be implemented in other fabricationprocesses involving greater numbers of metal layers. For example,distance between pixel signal lines and the topmost metal layer may beincreased by adding additional metal layers (more than four total metallayers) in which only jumpers to the topmost metal layer are formed inthe additional metal layers. In particular, a six-metal-layerfabrication process may be employed, in which the signal lines arefabricated using the Metal1 and Metal2 layers, the topmost metal layerof the floating gate structure is formed in the Metal6 layer, andjumpers to the topmost metal layer are formed in the Metal3, Metal4 andMetal5 layers, respectively (with associated vias between the metallayers). In another exemplary implementation based on a six-metal-layerfabrication process, the general pixel configuration shown in FIGS. 10,11A, and 12A-12L may be employed (signal lines on Metal2 and Metal 3layers), in which the topmost metal layer is formed in the Metal6 layerand jumpers are formed in the Metal4 and Metal5 layers, respectively.

In yet another aspect relating to reduced capacitance, a dimension “f”of the topmost metal layer 304 (and thus the ISFET sensitive area 178)may be reduced so as to reduce cross-capacitance between neighboringpixels. As may be observed in FIG. 11A-1 (and as discussed further belowin connection with other embodiments directed to well fabrication abovean ISFET array), the well 725 may be fabricated so as to have a taperedshape, such that a dimension “g” at the top of the well is smaller thanthe pixel pitch “e” but yet larger than a dimension “f” at the bottom ofthe well. Based on such tapering, the topmost metal layer 304 also maybe designed with the dimension “f” rather than the dimension “g” so asto provide for additional space between the top metal layers ofneighboring pixels. In some illustrative non-limiting implementations,for pixels having a dimension “e” on the order of 9 micrometers thedimension “f” may be on the order of 6 micrometers (as opposed to 7micrometers, as discussed above), and for pixels having a dimension “e”on the order of 5 micrometers the dimension “f” may be on the order of3.5 micrometers.

Thus, the pixel chip layout designs respectively shown in FIGS. 10, 11A,and 12A through 12L, and FIGS. 10-1, 11A-1, and 12-1A through 12-1L,illustrate that according to various embodiments FET devices of a sametype may be employed for all components of a pixel, and that allcomponents may be implemented in a single well. This dramaticallyreduces the area required for the pixel, thereby facilitating increasedpixel density in a given area.

In one exemplary implementation, the gate oxide 165 for the ISFET may befabricated to have a thickness on the order of approximately 75Angstroms, giving rise to a gate oxide capacitance per unit area C_(ox)of 4.5 fF/μm². Additionally, the polysilicon gate 164 may be fabricatedwith dimensions corresponding to a channel width W of 1.2 μm and achannel length L of from 0.35 to 0.6 μm (i.e., W/L ranging fromapproximately 2 to 3.5), and the doping of the region 160 may beselected such that the carrier mobility for the p-channel is 190 cm²/V·s(i.e., 1.9E10 μm²/V·s). From Eq. (2) above, this results in an ISFETtransconductance parameter β on the order of approximately 170 to 300μA/V². In other aspects of this exemplary implementation, the analogsupply voltage VDDA is 3.3 Volts, and VB1 and VB2 are biased so as toprovide a constant ISFET drain current I_(D); on the order of 5 μA (insome implementations, VB1 and VB2 may be adjusted to provide draincurrents from approximately 1 μA to 20 μA). Additionally, the MOSFET Q6(see bias/readout circuitry 110 j in FIG. 9) is sized to have a channelwidth to length ratio (e.g., W/L of approximately 50) such that thevoltage across Q6, given I_(Dj) of 5 μA, is 800 mV (i.e., V_(DSj)=800mV). From Eq. (3), based on these exemplary parameters, this providesfor pixel output voltages V_(Sj) over a range of approximately 0.5 to2.5 Volts for ISFET threshold voltage changes over a range ofapproximately 0 to 2 Volts.

With respect to the analyte-sensitive passivation layer 172 shown inFIG. 11A, in exemplary CMOS implementations the passivation layer may besignificantly sensitive to the concentration of various ion species,including hydrogen, and may include silicon nitride (Si₃N₄) and/orsilicon oxynitride (Si₂N₂O). In conventional CMOS processes, apassivation layer may be formed by one or more successive depositions ofthese materials, and is employed generally to treat or coat devices soas to protect against contamination and increase electrical stability.The material properties of silicon nitride and silicon oxynitride aresuch that a passivation layer comprising these materials providesscratch protection and serves as a significant barrier to the diffusionof water and sodium, which can cause device metallization to corrodeand/or device operation to become unstable. A passivation layerincluding silicon nitride and/or silicon oxynitride also providesion-sensitivity in ISFET devices, in that the passivation layer containssurface groups that may donate or accept protons from an analytesolution with which they are in contact, thereby altering the surfacepotential and the device threshold voltage V_(TH), as discussed above inconnection with FIGS. 1 and 2A.

For CMOS processes involving aluminum as the metal (which has a meltingpoint of approximately 650 degrees Celsius), a silicon nitride and/orsilicon oxynitride passivation layer generally is formed viaplasma-enhanced chemical vapor deposition (PECVD), in which a glowdischarge at 250-350 degrees Celsius ionizes the constituent gases thatform silicon nitride or silicon oxynitride, creating active species thatreact at the wafer surface to form a laminate of the respectivematerials. In one exemplary process, a passivation layer having athickness on the order of approximately 1.0 to 1.5 μm may be formed byan initial deposition of a thin layer of silicon oxynitride (on theorder of 0.2 to 0.4 μm) followed by a slighting thicker deposition ofsilicon oxynitride (on the order of 0.5 μm) and a final deposition ofsilicon nitride (on the order of 0.5 μm). Because of the low depositiontemperature involved in the PECVD process, the aluminum metallization isnot adversely affected.

However, Applicants have recognized and appreciated that while alow-temperature PECVD process provides adequate passivation forconventional CMOS devices, the low-temperature process results in agenerally low-density and somewhat porous passivation layer, which insome cases may adversely affect ISFET threshold voltage stability. Inparticular, during ISFET device operation, a low-density porouspassivation layer over time may absorb and become saturated with ionsfrom the solution, which may in turn cause an undesirable time-varyingdrift in the ISFETs threshold voltage V_(TH), making accuratemeasurements challenging.

In view of the foregoing, in one embodiment a CMOS process that usestungsten metal instead of aluminum may be employed to fabricate ISFETarrays according to the present disclosure. The high melting temperatureof Tungsten (above 3400 degrees Celsius) permits the use of a highertemperature low pressure chemical vapor deposition (LPCVD) process(e.g., approximately 700 to 800 degrees Celsius) for a silicon nitrideor silicon oxynitride passivation layer. The LPCVD process typicallyresults in significantly more dense and less porous films for thepassivation layer, thereby mitigating the potentially adverse effects ofion absorption from the analyte solution leading to ISFET thresholdvoltage drift.

In yet another embodiment in which an aluminum-based CMOS process isemployed to fabricate ISFET arrays according to the present disclosure,the passivation layer 172 shown in FIG. 11A may comprise additionaldepositions and/or materials beyond those typically employed in aconventional CMOS process. For example, the passivation layer 172 mayinclude initial low-temperature plasma-assisted depositions (PECVD) ofsilicon nitride and/or silicon oxynitride as discussed above; forpurposes of the present discussion, these conventional depositions areillustrated in FIG. 11A as a first portion 172A of the passivation layer172. In one embodiment, following the first portion 172A, one or moreadditional passivation materials are disposed to form at least a secondportion 172B to increase density and reduce porosity of (and absorptionby) the overall passivation layer 172. While one additional portion 172Bis shown primarily for purposes of illustration in FIG. 11A, it shouldbe appreciated that the disclosure is not limited in this respect, asthe overall passivation layer 172 may comprise two or more constituentportions, in which each portion may comprise one or morelayers/depositions of same or different materials, and respectiveportions may be configured similarly or differently.

Examples of materials suitable for the second portion 172B (or otheradditional portions) of the passivation layer 172 include, but are notlimited to, silicon nitride, silicon oxynitride, aluminum oxide (Al₂O₃),tantalum oxide (Ta₃O₅), tin oxide (SnO₂) and silicon dioxide (SiO₂). Inone aspect, the second portion 172B (or other additional portions) maybe deposited via a variety of relatively low-temperature processesincluding, but not limited to, RF sputtering, DC magnetron sputtering,thermal or e-beam evaporation, and ion-assisted depositions. In anotheraspect, a pre-sputtering etch process may be employed, prior todeposition of the second portion 172B, to remove any native oxideresiding on the first portion 172A (alternatively, a reducingenvironment, such as an elevated temperature hydrogen environment, maybe employed to remove native oxide residing on the first portion 172A).In yet another aspect, a thickness of the second portion 172B may be onthe order of approximately 0.04 μm to 0.06 μm (400 to 600 Angstroms) anda thickness of the first portion may be on the order of 1.0 to 1.5 μm,as discussed above. In some exemplary implementations, the first portion172A may include multiple layers of silicon oxynitride and siliconnitride having a combined thickness of 1.0 to 1.5 μm, and the secondportion 172B may include a single layer of either aluminum oxide ortantalum oxide having a thickness of approximately 400 to 600 Angstroms.Again, it should be appreciated that the foregoing exemplary thicknessesare provided primarily for purposes of illustration, and that thedisclosure is not limited in these respects.

It has been found according to the invention that hydrogen ion sensitivepassivation layers are also sensitive to other analytes including butnot limited to PPi and unincorporated nucleotide triphosphates. As anexample, a silicon nitride passivation layer is able to detect changesin the concentration of PPi and nucleotide triphosphates. The ability tomeasure the concentration change of these analytes using the samechemFET greatly facilitates the ability to sequence a nucleic acid usinga single array, thereby simplifying the sequencing method.

Thus it is to be understood that the chemFET arrays described herein maybe used to detect and/or measure various analytes and, by doing so, maymonitor a variety of reactions and/or interactions. It is also to beunderstood that the discussion herein relating to hydrogen ion detection(in the form of a pH change) is for the sake of convenience and brevityand that static or dynamic levels/concentrations of other analytes(including other ions) can be substituted for hydrogen in thesedescriptions. In particular, sufficiently fast concentration changes ofany one or more of various ion species present in the analyte may bedetected via the transient or dynamic response of a chemFET, asdiscussed above in connection with FIG. 2A. As also discussed above inconnection with the Site-Dissociation (or Site-Binding) model for theanalyte/passivation layer interface, it should be appreciated thatvarious parameters relating to the equilibrium reactions at theanalyte/passivation layer interface (e.g., rate constants for forwardand backward equilibrium reactions, total number of protondonor/acceptor sites per unit area on the passivation layer surface,intrinsic buffering capacity, pH at point of zero charge) are materialdependent properties and thus are affected by the choice of materialsemployed for the passivation layer.

The chemFETs, including ISFETs, described herein are capable ofdetecting any analyte that is itself capable of inducing a change inelectric field when in contact with or otherwise sensed or detected bythe chemFET surface. The analyte need not be charged in order to bedetected by the sensor. For example, depending on the embodiment, theanalyte may be positively charged (i.e., a cation), negatively charged(i.e., an anion), zwitterionic (i.e., capable of having two equal andopposite charges but being neutral overall), and polar yet neutral. Thislist is not intended as exhaustive as other analyte classes as well asspecies within each class will be readily contemplated by those ofordinary skill in the art based on the disclosure provided herein.

In the broadest sense of the invention, the passivation layer may or maynot be coated and the analyte may or may not interact directly with thepassivation layer. As an example, the passivation layer may be comprisedof silicon nitride and the analyte may be something other than hydrogenions. As a specific example, the passivation layer may be comprised ofsilicon nitride and the analyte may be PPi. In these instances, PPi isdetected directly (i.e., in the absence of PPi receptors attached to thepassivation layer either directly or indirectly).

If the analyte being detected is hydrogen (or alternatively hydroxide),then it is preferable to use weak buffers so that changes in eitherionic species can be detected at the passivation layer. If the analytebeing detected is something other than hydrogen (or hydroxide) but thereis some possibility of a pH change in the solution during the reactionor detection step, then it is preferable to use a strong buffer so thatchanges in pH do not interfere with the detection of the analyte. Abuffer is an ionic molecule (or a solution comprising an ionic molecule)that resists to varying extents changes in pH. Some buffers are able toneutralize acids or bases added to or generated in a solution, resultingin no effective pH change in the solution. It is to be understood thatany buffer is suitable provided it has a pKa in the desired range. Forsome embodiments, a suitable buffer is one that functions in about thepH range of 6 to 9, and more preferably 6.5 to 8.5. In otherembodiments, a suitable buffer is one that functions in about the pHrange of 7-10, including 8.5-9.5.

The strength of a buffer is a relative term since it depends on thenature, strength and concentration of the acid or base added to orgenerated in the solution of interest. A weak buffer is a buffer thatallows detection (and therefore is not able to otherwise control) pHchanges of about at least +/−0.005, about at least +/−0.01, about atleast +/−0.015, about at least +/−0.02, about at least +/−0.03, about atleast +/−0.04, about at least +/−0.05, about at least +/−0.10, about atleast +/−0.15, about at least +/−0.20, about at least +/−0.25, about atleast +/−0.30, about at least +/−0.35, about at least +/−0.45, about atleast +/−0.50, or more. In some embodiments, the pH change is on theorder of about 0.005 (e.g., per nucleotide incorporation) and ispreferably an increase in pH. A strong buffer is a buffer that controlspH changes of about at least +/−0.005, about at least +/−0.01, about atleast +/−0.015, about at least +/−0.02, about at least +/−0.03, about atleast +/−0.04, about at least +/−0.05, about at least +/−0.10, about atleast +/−0.15, about at least +/−0.20, about at least +/−0.25, about atleast +/−0.30, about at least +/−0.35, about at least +/−0.45, about atleast +/−0.50, or more. Buffer strength can be varied by varying theconcentration of the buffer species itself. Thus low concentrationbuffers can be low strength buffers. Examples include those having lessthan 1 mM (e.g., 50-100 μM) buffer species. A non-limiting example of aweak buffer suitable for the sequencing reactions described hereinwherein pH change is the readout is 0.1 mM Tris or Tricine. Examples ofsuitable weak buffers are provided in the Examples and are also known inthe art. Higher concentration buffers can be stronger buffers. Examplesinclude those having 1-25 mM buffer species. A non-limiting example of astrong buffer suitable for the sequencing reactions described hereinwherein PPi and/or nucleotide triphosphates are read directly is 1, 5 or25 mM (or more) Tris or Tricine. One of ordinary skill in the art willbe able to determine the optimal buffer for use in the reactions anddetection methods encompassed by the invention.

In some embodiments, the passivation layer and/or the layers and/ormolecules coated thereon dictate the analyte specificity of the arrayreadout.

Detection of hydrogen ions (in the form of pH), and other analytes asdetermined by the invention, can be carried out using a passivationlayer made of silicon nitride (Si₃N₄), silicon oxynitride (Si₂N₂O),silicon oxide (SiO₂), aluminum oxide (Al₂O₃), tantalum pentoxide(Ta₂O₅), tin oxide or stannic oxide (SnO₂), and the like.

The passivation layer can also detect other ion species directlyincluding but not limited to calcium, potassium, sodium, iodide,magnesium, chloride, lithium, lead, silver, cadmium, nitrate, phosphate,dihydrogen phosphate, and the like.

In some embodiments, the passivation layer is coated with a receptor forthe analyte of interest. Preferably, the receptor binds selectively tothe analyte of interest or in some instances to a class of agents towhich the analyte belongs. As used herein, a receptor that bindsselectively to an analyte is a molecule that binds preferentially tothat analyte (i.e., its binding affinity for that analyte is greaterthan its binding affinity for any other analyte). Its binding affinityfor the analyte of interest may be 2-fold, 3-fold, 4-fold, 5-fold,6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold,30-fold, 40-fold, 50-fold, 100-fold or more than its binding affinityfor any other analyte. In addition to its relative binding affinity, thereceptor must also have an absolute binding affinity that issufficiently high to efficiently bind the analyte of interest (i.e., itmust have a sufficient sensitivity). Receptors having binding affinitiesin the picomolar to micromolar range are suitable. Preferably suchinteractions are reversible.

The receptor may be of any nature (e.g., chemical, nucleic acid,peptide, lipid, combinations thereof and the like). In such embodiments,the analyte too may be of any nature provided there exists a receptorthat binds to it selectively and in some instances specifically. It isto be understood however that the invention further contemplatesdetection of analytes in the absence of a receptor. An example of thisis the detection of PPi and Pi by the passivation layer in the absenceof PPi or Pi receptors.

In one aspect, the invention contemplates receptors that are ionophores.As used herein, an ionophore is a molecule that binds selectively to anionic species, whether anion or cation. In the context of the invention,the ionophore is the receptor and the ion to which it binds is theanalyte. Ionophores of the invention include art-recognized carrierionophores (i.e., small lipid-soluble molecules that bind to aparticular ion) derived from microorganisms. Various ionophores arecommercially available from sources such as Calbiochem.

Detection of some ions can be accomplished through the use of thepassivation layer itself or through the use of receptors coated onto thepassivation layer. For example, potassium can be detected selectivelyusing polysiloxane, valinomycin, or salinomycin; sodium can be detectedselectively using monensin, nystatin, or SQI-Pr; calcium can be detectedselectively using ionomycin, calcimycine (A23187), or CA 1001 (ETH1001).

Receptors able to bind more than one ion can also be used in someinstances. For example, beauvericin can be used to detect calcium and/orbarium ions, nigericin can be used to detect potassium, hydrogen and/orlead ions, and gramicidin can be used to detect hydrogen, sodium and/orpotassium ions. One of ordinary skill in the art will recognize thatthese compounds can be used in applications in which single ionspecificity is not required or in which it is unlikely (or impossible)that other ions which the compounds bind will be present or generated.Similarly, receptors that bind multiple species of a particular genusmay also be useful in some embodiments including those in which only onespecies within the genus will be present or in which the method does notrequire distinction between species.

As another example, receptors for neurotoxins are described in SimonianElectroanalysis 2004, 16: 1896-1906.

In other embodiments, including but not limited to nucleic acidsequencing applications, receptors that bind selectively to PPi can beused. Examples of PPi receptors include those compounds shown in FIGS.11B(1)-(3) (compounds 1-10). Compound 1 is described in Angew, Chem Int(Ed 2004) 43:4777-4780 and US 2005/0119497 A1 and is referred to asp-naphthyl-bis[(bis(2-pyridylmethyl)amino)methyl]phenol. Compound 2 isdescribed in J Am Chem Soc 2003 125:7752-7753 and US 2005/0119497 A1 andis referred to asp-(p-nitrophenylazo)-bis[(bis(2-pyridylmethyl-1)amino)methyl]phenol (orits dinuclear Zn complex). Synthesis schemes for compounds 1 and 2 areshown provided in US 2005/0119497 A1. Compound 3 is described in by Leeet al. Organic Letters 2007 9(2):243-246, and Sensors and Actuators B1995 29:324-327. Compound 4 is described in Angew, Chem Int (Ed 2002)41(20):3811-3814. Exemplary syntheses for compounds 7, 8 and 9 are shownin FIGS. 11C(1)-(3). Compound 5 is described in WO 2007/002204 and isreferred to therein as bis-Zn²⁺-dipicolylamine (Zn²⁺-DPA). Compound 6 isillustrated in FIG. 11B(3) bound to PPi. (McDonough et al. Chem. Commun.2006 2971-2973.) Attachment of compound 7 to a metal oxide surface isshown in FIG. 11E.

Receptors may be attached to the passivation layer covalently ornon-covalently. Covalent attachment of a receptor to the passivationlayer may be direct or indirect (e.g., through a linker). FIGS. 11D(1)and (2) illustrate the use of silanol chemistry to covalently bindreceptors to the passivation layer. Receptors may be immobilized on thepassivation layer using for example aliphatic primary amines (bottomleft panel) or aryl isothiocyanates (bottom right panel). In these andother embodiments, the passivation layer which itself may be comprisedof silicon nitride, aluminum oxide, silicon oxide, tantalum pentoxide,or the like, is bonded to a silanation layer via its reactive surfacegroups. For greater detail on silanol chemistry for covalent attachmentto the FET surface, reference can be made to at least the followingpublications: for silicon nitride, see Sensors and Actuators B 199529:324-327, Jpn J Appl Phys 1999 38:3912-3917 and Langmuir 200521:395-402; for silicon oxide, see Protein Sci 1995 4:2532-2544 and AmBiotechnol Lab 2002 20(7):16-18; and for aluminum oxide, see Colloidsand Surfaces 1992 63:1-9, Sensors and Actuators B 2003 89:40-47, andBioconjugate Chem 1997 8:424-433. The receptor is then conjugated to thesilanation layer reactive groups. This latter binding can occur directlyor indirectly through the use of a bifunctional linker, as illustratedin FIGS. 11D(1) and (2).

A bifunctional linker is a compound having at least two reactive groupsto which two entities may be bound. In some instances, the reactivegroups are located at opposite ends of the linker. In some embodiments,the bifunctional linker is a universal bifunctional linker such as thatshown in FIGS. 11D(1) and (2). A universal linker is a linker that canbe used to link a variety of entities. It should be understood that thechemistries shown in FIGS. 11D(1) and (2) are meant to be illustrativeand not limiting.

The bifunctional linker may be a homo-bifunctional linker or ahetero-bifunctional linker, depending upon the nature of the moleculesto be conjugated. Homo-bifunctional linkers have two identical reactivegroups. Hetero-bifunctional linkers are have two different reactivegroups. Various types of commercially available linkers are reactivewith one or more of the following groups: primary amines, secondaryamines, sulphydryls, carboxyls, carbonyls and carbohydrates. Examples ofamine-specific linkers are bis(sulfosuccinimidyl) suberate,bis[2-(succinimidooxycarbonyloxy)ethyl]sulfone, disuccinimidyl suberate,disuccinimidyl tartarate, dimethyl adipimate.2HCl, dimethylpimelimidate.2HCl, dimethyl suberimidate.2HCl, and ethyleneglycolbis-[succinimidyl-[succinate]]. Linkers reactive with sulfhydrylgroups include bismaleimidohexane,1,4-di-[3′-(2′-pyridyldithio)-propionamido)]butane,1-[p-azidosalicylamido]-4-[iodoacetamido]butane, andN-[4-(p-azidosalicylamido) butyl]-3′-[2′-pyridyldithio]propionamide.Linkers preferentially reactive with carbohydrates include azidobenzoylhydrazine. Linkers preferentially reactive with carboxyl groups include4-[p-azidosalicylamido]butylamine.

Heterobifunctional linkers that react with amines and sulfhydrylsinclude N-succinimidyl-3-[2-pyridyldithio]propionate,succinimidyl[4-iodoacetyl]aminobenzoate, succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate,m-maleimidobenzoyl-N-hydroxysuccinimide ester, sulfosuccinimidyl6-[3-[2-pyridyldithio]propionamido]hexanoate, and sulfosuccinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate. Heterobifunctionallinkers that react with carboxyl and amine groups include1-ethyl-3-[3-dimethylaminopropyl]-carbodiimide hydrochloride.Heterobifunctional linkers that react with carbohydrates and sulfhydrylsinclude 4-[N-maleimidomethyl]-cyclohexane-1-carboxylhydrazide.2HCl,4-(4-N-maleimidophenyl)-butyric acid hydrazide.2HCl, and3-[2-pyridyldithio]propionyl hydrazide.

Alternatively, receptors may be non-covalently coated onto thepassivation layer. Non-covalent deposition of the receptor onto thepassivation layer may involve the use of a polymer matrix. The polymermay be naturally occurring or non-naturally occurring and may be of anytype including but not limited to nucleic acid (e.g., DNA, RNA, PNA,LNA, and the like, or mimics, derivatives, or combinations thereof),amino acid (e.g., peptides, proteins (native or denatured), and thelike, or mimics, derivatives, or combinations thereof, lipids,polysaccharides, and functionalized block copolymers. The receptor maybe adsorbed onto and/or entrapped within the polymer matrix. The natureof the polymer will depend on the nature of the receptor being usedand/or analyte being detected.

Alternatively, the receptor may be covalently conjugated or crosslinkedto the polymer (e.g., it may be “grafted” onto a functionalizedpolymer).

An example of a suitable peptide polymer is poly-lysine (e.g.,poly-L-lysine). Examples of other polymers include block copolymers thatcomprise polyethylene glycol (PEG), polyamides, polycarbonates,polyalkylenes, polyalkylene glycols, polyalkylene oxides, polyalkyleneterepthalates, polyvinyl alcohols, polyvinyl ethers, polyvinyl esters,polyvinyl halides, polyvinylpyrrolidone, polyglycolides, polysiloxanes,polyurethanes, alkyl cellulose, hydroxyalkyl celluloses, celluloseethers, cellulose esters, nitrocelluloses, polymers of acrylic andmethacrylic esters, methyl cellulose, ethyl cellulose, hydroxypropylcellulose, hydroxypropyl methyl cellulose, hydroxybutyl methylcellulose, cellulose acetate, cellulose propionate, cellulose acetatebutyrate, cellulose acetate phthalate, carboxylethyl cellulose,cellulose triacetate, cellulose sulphate sodium salt, poly(methylmethacrylate), poly(ethyl methacrylate), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), poly(octadecyl acrylate), polyethylene, polypropylene,poly(ethylene glycol), poly(ethylene oxide), poly(ethyleneterephthalate), poly(vinyl alcohols), polyvinyl acetate, polyvinylchloride, polystyrene, polyhyaluronic acids, casein, gelatin, glutin,polyanhydrides, polyacrylic acid, alginate, chitosan, poly(methylmethacrylates), poly(ethyl methacrylates), poly(butylmethacrylate),poly(isobutyl methacrylate), poly(hexylmethacrylate), poly(isodecylmethacrylate), poly(lauryl methacrylate), poly(phenyl methacrylate),poly(methyl acrylate), poly(isopropyl acrylate), poly(isobutylacrylate), and poly(octadecyl acrylate), poly(lactide-glycolide),copolyoxalates, polycaprolactones, polyesteramides, polyorthoesters,polyhydroxybutyric acid, polyanhydrides,poly(styrene-b-isobutylene-b-styrene) (SIBS) block copolymer, ethylenevinyl acetate, poly(meth)acrylic acid, polymers of lactic acid andglycolic acid, polyanhydrides, poly(ortho)esters, polyurethanes,poly(butic acid), poly(valeric acid), and poly(lactide-cocaprolactone),and natural polymers such as alginate and other polysaccharidesincluding dextran and cellulose, collagen, albumin and other hydrophilicproteins, zein and other prolamines and hydrophobic proteins, copolymersand mixtures thereof, and chemical derivatives thereof includingsubstitutions and/or additions of chemical groups, for example, alkyl,alkylene, hydroxylations, oxidations, and other modifications routinelymade by those skilled in the art.

Another issue that relates to ISFET threshold voltage stability and/orpredictability involves trapped charge that may accumulate (especially)on metal layers of CMOS-fabricated devices as a result of variousprocessing activities during or following array fabrication (e.g.,back-end-of-line processing such as plasma metal etching, wafercleaning, dicing, packaging, handling, etc.). In particular, withreference to FIG. 11A, trapped charge may in some instances accumulateon one or more of the various conductors 304, 306, 308, 312, 316, 320,326, 338, and 164 constituting the ISFETs floating gate structure 170.This phenomenon also is referred to in the relevant literature as the“antenna effect.”

One opportunity for trapped charge to accumulate includes plasma etchingof the topmost metal layer 304. Applicants have recognized andappreciated that other opportunities for charge to accumulate on one ormore conductors of the floating gate structure or other portions of theFETs includes wafer dicing, during which the abrasive process of adicing saw cutting through a wafer generates static electricity, and/orvarious post-processing wafer handling/packaging steps, such asdie-to-package wire bonding, where in some cases automated machinerythat handles/transports wafers may be sources of electrostatic discharge(ESD) to conductors of the floating gate structure. If there is noconnection to the silicon substrate (or other semi-conductor substrate)to provide an electrical path to bleed off such charge accumulation,charge may build up to the point of causing undesirable changes ordamage to the gate oxide 165 (e.g., charge injection into the oxide, orlow-level oxide breakdown to the underlying substrate). Trapped chargein the gate oxide or at the gate oxide-semiconductor interface in turncan cause undesirable and/or unpredictable variations in ISFET operationand performance, such as fluctuations in threshold voltage.

In view of the foregoing, other inventive embodiments of the presentdisclosure are directed to methods and apparatus for improving ISFETperformance by reducing trapped charge or mitigating the antenna effect.In one embodiment, trapped charge may be reduced after a sensor arrayhas been fabricated, while in other embodiments the fabrication processitself may be modified to reduce trapped charge that could be induced bysome conventional process steps. In yet other embodiments, both “duringfabrication” and “post fabrication” techniques may be employed incombination to reduce trapped charge and thereby improve ISFETperformance.

With respect to alterations to the fabrication process itself to reducetrapped charge, in one embodiment the thickness of the gate oxide 165shown in FIG. 11A may be particularly selected so as to facilitatebleeding of accumulated charge to the substrate; in particular, athinner gate oxide may allow a sufficient amount of built-up charge topass through the gate oxide to the substrate below without becomingtrapped. In another embodiment based on this concept, a pixel may bedesigned to include an additional “sacrificial” device, i.e., anothertransistor having a thinner gate oxide than the gate oxide 165 of theISFET. The floating gate structure of the ISFET may then be coupled tothe gate of the sacrificial device such that it serves as a “chargebleed-off transistor.” Of course, it should be appreciated that sometrade-offs for including such a sacrificial device include an increasein pixel size and complexity.

In another embodiment, the topmost metal layer 304 of the ISFETsfloating gate structure 170 shown in FIG. 11A may be capped with adielectric prior to plasma etching to mitigate trapped charge. Asdiscussed above, charge accumulated on the floating gate structure mayin some cases be coupled from the plasma being used for metal etching.Typically, a photoresist is applied over the metal to be etched and thenpatterned based on the desired geometry for the underlying metal. In oneexemplary implementation, a capping dielectric layer (e.g., an oxide)may be deposited over the metal to be etched, prior to the applicationof the photoresist, to provide an additional barrier on the metalsurface against charge from the plasma etching process. In one aspect,the capping dielectric layer may remain behind and form a portion of thepassivation layer 172.

In yet another embodiment, the metal etch process for the topmost metallayer 304 may be modified to include wet chemistry or ion-beam millingrather than plasma etching. For example, the metal layer 304 could beetched using an aqueous chemistry selective to the underlying dielectric(e.g., see website for Transene relating to aluminum, which is herebyincorporated herein by reference). Another alternative approach employsion-milling rather than plasma etching for the metal layer 304.Ion-milling is commonly used to etch materials that cannot be readilyremoved using conventional plasma or wet chemistries. The ion-millingprocess does not employ an oscillating electric field as does a plasma,so that charge build-up does not occur in the metal layer(s). Yetanother metal etch alternative involves optimizing the plasma conditionsso as to reduce the etch rate (i.e. less power density).

In yet another embodiment, architecture changes may be made to the metallayer to facilitate complete electrical isolation during definition ofthe floating gate. In one aspect, designing the metal stack-up so thatthe large area ISFET floating gate is not connected to anything duringits final definition may require a subsequent metal layer serving as a“jumper” to realize the electrical connection to the floating gate ofthe transistor. This “jumper” connection scheme prevents charge flowfrom the large floating gate to the transistor. This method may beimplemented as follows (M=metal layer): i) M1 contacting Poly gateelectrode; ii) M2 contacting M1; iii) M3 defines floating gate andseparately connects to M2 with isolated island; iv) M4 jumper, havingvery small area being etched over the isolated islands and connectionsto floating gate M3, connects the M3 floating gate to the M1/M2/M3 stackconnected to the Poly gate immediately over the transistor active area;and v) M3 to M4 interlayer dielectric is removed only over the floatinggate so as to expose the bare M3 floating gate. In the method outlinedimmediately above, step v) need not be done, as the ISFET architectureaccording to some embodiments discussed above leaves the M4 passivationin place over the M4 floating gate. In one aspect, removal maynonetheless improve ISFET performance in other ways (i.e. sensitivity).In any case, the final sensitive passivation layer may be a thinsputter-deposited ion-sensitive metal-oxide layer. It should beappreciated that the over-layer jumpered architecture discussed abovemay be implemented in the standard CMOS fabrication flow to allow any ofthe first three metal layers to be used as the floating gates (i.e. M1,M2 or M3).

With respect to post-fabrication processes to reduce trapped charge, inone embodiment a “forming gas anneal” may be employed as apost-fabrication process to mitigate potentially adverse effects oftrapped charge. In a forming gas anneal, CMOS-fabricated ISFET devicesare heated in a hydrogen and nitrogen gas mixture. The hydrogen gas inthe mixture diffuses into the gate oxide 165 and neutralizes certainforms of trapped charges. In one aspect, the forming gas anneal need notnecessarily remove all gate oxide damage that may result from trappedcharges; rather, in some cases, a partial neutralization of some trappedcharge is sufficient to significantly improve ISFET performance. Inexemplary annealing processes according to the present disclosure,ISFETs may be heated for approximately 30 to 60 minutes at approximately400 to 425 degrees Celsius in a hydrogen/nitrogen mixture that includes10% to 15% hydrogen. In one particular implementation, annealing at 425degrees Celsius at 30 minutes in a hydrogen/nitrogen mixture thatincludes 10% hydrogen is observed to be particularly effective atimproving ISFET performance. For aluminum CMOS processes, thetemperature of the anneal should be kept at or below 450 degrees Celsiusto avoid damaging the aluminum metallurgy. In another aspect of anannealing process according to the present disclosure, the forming gasanneal is performed after wafers of fabricated ISFET arrays are diced,so as to ensure that damage due to trapped charge induced by the dicingprocess itself, and/or other pre-dicing processing steps (e.g., plasmaetching of metals) may be effectively ameliorated. In yet anotheraspect, the forming gas anneal may be performed after die-to-packagewirebonding to similarly ameliorate damage due to trapped charge. Atthis point in the assembly process, a diced array chip is typically in aheat and chemical resistant ceramic package, and low-tolerancewirebonding procedures as well as heat-resistant die-to-packageadhesives may be employed to withstand the annealing procedure. Thus, inone exemplary embodiment, the invention encompasses a method formanufacturing an array of FETs, each having or coupled to a floatinggate having a trapped charge of zero or substantially zero comprising:fabricating a plurality of FETs in a common semiconductor substrate,each of a plurality of which is coupled to a floating gate; applying aforming gas anneal to the semiconductor prior to a dicing step; dicingthe semiconductor; and applying a forming gas anneal to thesemiconductor after the dicing step. Preferably, the semiconductorsubstrate comprises at least 100,000 FETs. Preferably, the plurality ofFETs are chemFETs. The method may further comprise depositing apassivation layer on the semiconductor, depositing a polymeric, glass,ion-reactively etchable or photodefineable material layer on thepassivation layer and etching the polymeric, glass ion-reactivelyetchable or photodefineable material to form an array of reactionchambers in the glass layer.

In yet other processes for mitigating potentially adverse effects oftrapped charge according to embodiments of the present disclosure, avariety of “electrostatic discharge (ESD)-sensitive protocols” may beadopted during any of a variety of wafer post-fabricationhandling/packaging steps. For example, in one exemplary process,anti-static dicing tape may be employed to hold wafer substrates inplace (e.g., during the dicing process). Also, although high-resistivity(e.g., 10 MΩ) deionized water conventionally is employed in connectionwith cooling of dicing saws, according to one embodiment of the presentdisclosure less resistive/more conductive water may be employed for thispurpose to facilitate charge conduction via the water; for example,deionized water may be treated with carbon dioxide to lower resistivityand improve conduction of charge arising from the dicing process.Furthermore, conductive and grounded die-ejection tools may be usedduring various wafer dicing/handling/packaging steps, again to provideeffective conduction paths for charge generated during any of thesesteps, and thereby reduce opportunities for charge to accumulate on oneor more conductors of the floating gate structure of respective ISFETsof an array.

In yet another embodiment involving a post-fabrication process to reducetrapped charge, the gate oxide region of an ISFET may be irradiated withUV radiation. With reference again to FIG. 11A, in one exemplaryimplementation based on this embodiment, an optional hole or window 302is included during fabrication of an ISFET array in the top metal layer304 of each pixel of the array, proximate to the ISFET floating gatestructure. This window is intended to allow UV radiation, whengenerated, to enter the ISFETs gate region; in particular, the variouslayers of the pixel 105 ₁, as shown in FIGS. 11 and 12 A-L, areconfigured such that UV radiation entering the window 302 may impinge inan essentially unobstructed manner upon the area proximate to thepolysilicon gate 164 and the gate oxide 165.

To facilitate a UV irradiation process to reduce trapped charge,Applicants have recognized and appreciated that materials other thansilicon nitride and silicon oxynitride generally need to be employed inthe passivation layer 172 shown in FIG. 11A, as silicon nitride andsilicon oxynitride significantly absorb UV radiation. In view of theforegoing, these materials need to be substituted with others that areappreciably transparent to UV radiation, examples of which include, butare not limited to, phosphosilicate glass (PSG) and boron-dopedphosphosilicate glass (BPSG). PSG and BPSG, however, are not imperviousto hydrogen and hydroxyl ions; accordingly, to be employed in apassivation layer of an ISFET designed for pH sensitivity, PSG and BPSGmay be used together with an ion-impervious material that is alsosignificantly transparent to UV radiation, such as aluminum oxide(Al₂O₃), to form the passivation layer. For example, with referenceagain to FIG. 11A, PSG or BPSG may be employed as a substitute forsilicon nitride or silicon oxynitride in the first portion 172A of thepassivation layer 172, and a thin layer (e.g., 400 to 600 Angstroms) ofaluminum oxide may be employed in the second portion 172B of thepassivation layer 172 (e.g., the aluminum oxide may be deposited using apost-CMOS lift-off lithography process).

In another aspect of an embodiment involving UV irradiation, each ISFETof a sensor array must be appropriately biased during a UV irradiationprocess to facilitate reduction of trapped charge. In particular, highenergy photons from the UV irradiation, impinging upon the bulk siliconregion 160 in which the ISFET conducting channel is formed, createelectron-hole pairs which facilitate neutralization of trapped charge inthe gate oxide as current flows through the ISFETs conducting channel.To this end, an array controller, discussed further below in connectionwith FIG. 17, generates appropriate signals for biasing the ISFETs ofthe array during a UV irradiation process. In particular, with referenceagain to FIG. 9, each of the signals RowSel₁ through RowSel_(n) isgenerated so as to enable/select (i.e., turn on) all rows of the sensorarray at the same time and thereby couple all of the ISFETs of the arrayto respective controllable current sources 106 _(j) in each column. Withall pixels of each column simultaneously selected, the current from thecurrent source 106 of a given column is shared by all pixels of thecolumn. The column amplifiers 107A and 107B are disabled by removing thebias voltage VB4, and at the same time the output of the amplifier 107B,connected to the drain of each ISFET in a given column, is grounded viaa switch responsive to a control signal “UV.” Also, the common bodyvoltage V_(BODY) for all ISFETs of the array is coupled to electricalground (i.e., V_(BODY)=0 Volts) (as discussed above, during normaloperation of the array, the body bias voltage V_(BODY) is coupled to thehighest voltage potential available to the array, e.g., VDDA). In oneexemplary procedure, the bias voltage VB1 for all of the controllablecurrent sources 106 is set such that each pixel's ISFET conductsapproximately 1 μA of current. With the ISFET array thusly biased, thearray then is irradiated with a sufficient dose of UV radiation (e.g.,from an EPROM eraser generating approximately 20 milliWatts/cm² ofradiation at a distance of approximately one inch from the array forapproximately 1 hour). After irradiation, the array may be allowed torest and stabilize over several hours before use for measurements ofchemical properties such as ion concentration.

Utilizing at least one of the above-described techniques for reducingtrapped charge, we have been able to fabricate FETs floating gateshaving a trapped charge of zero or substantially zero. Thus, in someembodiments, an aspect of the invention encompasses a floating gatehaving a surface area of about 4 μm² to about 50 μm² having baselinethreshold voltage and preferably a trapped charge of zero orsubstantially zero. Preferably the FETs are chemFETs. The trapped chargeshould be kept to a level that does not cause appreciable variationsfrom FET to FET across the array, as that would limit the dynamic rangeof the devices, consistency of measurements, and otherwise adverselyaffect performance.

FIG. 13 illustrates a block diagram of an exemplary CMOS IC chipimplementation of an ISFET sensor array 100 based on the column andpixel designs discussed above in connection with FIGS. 9-12, accordingto one embodiment of the present disclosure. In one aspect of thisembodiment, the array 100 includes 512 columns 102 ₁ through 102 ₅₁₂with corresponding column bias/readout circuitry 110 ₁ through 110 ₅₁₂(one for each column, as shown in FIG. 9), wherein each column includes512 geometrically square pixels 105 ₁ through 105 ₅₁₂, each having asize of approximately 9 micrometers by 9 micrometers (i.e., the array is512 columns by 512 rows). In another aspect, the entire array (includingpixels together with associated row and column select circuitry andcolumn bias/readout circuitry) may be fabricated on a semiconductor dieas an application specific integrated circuit (ASIC) having dimensionsof approximately 7 millimeters by 7 millimeters. While an array of 512by 512 pixels is shown in the embodiment of FIG. 13, it should beappreciated that arrays may be implemented with different numbers ofrows and columns and different pixel sizes according to otherembodiments, as discussed further below in connection with FIGS. 19-23.

Also, as discussed above, it should be appreciated that arrays accordingto various embodiments of the present invention may be fabricatedaccording to conventional CMOS fabrications techniques, as well asmodified CMOS fabrication techniques (e.g., to facilitate realization ofvarious functional aspects of the chemFET arrays discussed herein, suchas additional deposition of passivation materials, process steps tomitigate trapped charge, etc.) and other semiconductor fabricationtechniques beyond those conventionally employed in CMOS fabrication.Additionally, various lithography techniques may be employed as part ofan array fabrication process. For example, in one exemplaryimplementation, a lithography technique may be employed in whichappropriately designed blocks are “stitched” together by overlapping theedges of a step and repeat lithography exposures on a wafer substrate byapproximately 0.2 micrometers. In a single exposure, the maximum diesize typically is approximately 21 millimeters by 21 millimeters. Byselectively exposing different blocks (sides, top & bottoms, core, etc.)very large chips can be defined on a wafer (up to a maximum, in theextreme, of one chip per wafer, commonly referred to as “wafer scaleintegration”).

In one aspect of the array 100 shown in FIG. 13, the first and last twocolumns 102 ₁, 102 ₂, 102 ₅₁₁ and 102 ₅₁₂, as well as the first twopixels 105 ₁ and 105 ₂ and the last two pixels 105 ₅₁₁ and 105 ₅₁₂ ofeach of the columns 102 ₃ through 102 ₅₁₀ (e.g., two rows and columns ofpixels around a perimeter of the array) may be configured as “reference”or “dummy” pixels 103. With reference to FIG. 11A, for the dummy pixelsof an array, the topmost metal layer 304, of each dummy pixel's ISFET(coupled ultimately to the ISFETs polysilicon gate 164) is tied to thesame metal layer of other dummy pixels and is made accessible as aterminal of the chip, which in turn may be coupled to a referencevoltage VREF. As discussed above in connection with FIG. 9, thereference voltage VREF also may be applied to the bias/readout circuitryof respective columns of the array. In some exemplary implementationsdiscussed further below, preliminary test/evaluation data may beacquired from the array based on applying the reference voltage VREF andselecting and reading out dummy pixels, and/or reading out columns basedon the direct application of VREF to respective column buffers (e.g.,via the CAL signal), to facilitate offset determination (e.g.,pixel-to-pixel and column-to-column variances) and array calibration.

In yet another implementation of an array similar to that shown in FIG.13, rather than reserving the first and last two columns of 512 columnsand the first and last two pixels of each column of 512 pixels asreference pixels, the array may be fabricated to include an additionaltwo rows/columns of reference pixels surrounding a perimeter of a 512 by512 region of active pixels, such that the total size of the array interms of actual pixels is 516 by 516 pixels. As arrays of various sizesand configurations are contemplated by the present disclosure, it shouldbe appreciated that the foregoing concept may be applied to any of theother array embodiments discussed herein. For purposes of the discussionimmediately below regarding the exemplary array 100 shown in FIG. 13, atotal pixel count for the array of 512 by 512 pixels is considered.

In FIG. 13, various power supply and bias voltages required for arrayoperation (as discussed above in connection with FIG. 9) are provided tothe array via electrical connections (e.g., pins, metal pads) andlabeled for simplicity in block 195 as “supply and bias connections.”The array 100 of FIG. 13 also includes a row select shift register 192,two sets of column select shift registers 194 _(1,2) and two outputdrivers 198 ₁ and 198 ₂ to provide two parallel array output signals,Vout1 and Vout2, representing sensor measurements (i.e., collections ofindividual output signals generated by respective ISFETs of the array).The various power supply and bias voltages, control signals for the rowand column shift registers, and control signals for the columnbias/readout circuitry shown in FIG. 13 are provided by an arraycontroller, as discussed further below in connection with FIG. 17, whichalso reads the array output signals Vout1 and Vout2 (and other optionalstatus/diagnostic signals) from the array 100. In another aspect of thearray embodiment shown in FIG. 13, configuring the array such thatmultiple regions (e.g., multiple columns) of the array may be read atthe same time via multiple parallel array output signals (e.g., Vout1and Vout2) facilitates increased data acquisition rates, as discussedfurther below in connection with FIGS. 17 and 18. While FIG. 13illustrates an array having two column select registers and parallelarray output signals Vout1 and Vout2 to acquire data simultaneously fromtwo columns at a time, it should be appreciated that, in otherembodiments, arrays according to the present disclosure may beconfigured to have only one measurement signal output, or more than twomeasurement signal outputs; in particular, as discussed further below inconnection with FIGS. 19-23, more dense arrays according to otherinventive embodiments may be configured to have four our more parallelmeasurement signal outputs and simultaneously enable different regionsof the array to provide data via the four or more outputs.

FIG. 14 illustrates the row select shift register 192, FIG. 15illustrates one of the column select shift registers 194 ₂ and FIG. 16illustrates one of the output drivers 198 ₂ of the array 100 shown inFIG. 13, according to one exemplary implementation. As shown in FIGS. 14and 15, the row and column select shift registers are implemented as aseries of D-type flip-flops coupled to a digital circuitry positivesupply voltage VDDD and a digital supply ground VSSD. In the row andcolumn shift registers, a data signal is applied to a D-input of firstflip-flop in each series and a clock signal is applied simultaneously toa clock input of all of the flip-flops in the series. For eachflip-flop, a “Q” output reproduces the state of the D-input upon atransition (e.g., falling edge) of the clock signal. With reference toFIG. 14, the row select shift register 192 includes 512 D-typeflip-flops, in which a first flip-flop 193 receives a vertical datasignal DV and all flip-flops receive a vertical clock signal CV. A “Q”output of the first flip-flop 193 provides the first row select signalRowSel₁ and is coupled to the D-input of the next flip-flop in theseries. The Q outputs of successive flip-flops are coupled to theD-inputs of the next flip-flop in the series and provide the row selectsignals RowSel₂ through RowSel₅₁₂ with successive falling edgetransitions of the vertical clock signal CV, as discussed further belowin connection with FIG. 18. The last row select signal RowSel₅₁₂ alsomay be taken as an optional output of the array 100 as the signal LSTV(Last STage Vertical), which provides an indication (e.g., fordiagnostic purposes) that the last row of the array has been selected.While not shown explicitly in FIG. 14, each of the row select signalsRowSel₁ through RowSel₅₁₂ is applied to a corresponding inverter, theoutput of which is used to enable a given pixel in each column (asillustrated in FIG. 9 by the signals RowSel₁ through RowSel_(n) ).

Regarding the column select shift registers 194 ₁ and 194 ₂, these areimplemented in a manner similar to that of the row select shiftregisters, with each column select shift register comprising 256series-connected flip-flops and responsible for enabling readout fromeither the odd columns of the array or the even columns of the array.For example, FIG. 15 illustrates the column select shift register 194 ₂,which is configured to enable readout from all of the even numberedcolumns of the array in succession via the column select signalsColSel₂, ColSel₄, . . . ColSel₅₁₂, whereas another column select shiftregister 194 ₁ is configured to enable readout from all of the oddnumbered columns of the array in succession (via column select signalsColSel₁, ColSel₃, . . . ColSel₅₁₁). Both column select shift registersare controlled simultaneously by the horizontal data signal DH and thehorizontal clock signal CH to provide the respective column selectsignals, as discussed further below in connection with FIG. 18. As shownin FIG. 15, the last column select signal ColSel₅₁₂ also may be taken asan optional output of the array 100 as the signal LSTH (Last STageHorizontal), which provides an indication (e.g., for diagnosticpurposes) that the last column of the array has been selected.

With reference again for the moment to FIG. 7, Applicants haverecognized and appreciated that an implementation for array row andcolumn selection based on shift registers, as discussed above inconnection with FIGS. 13-15, is a significant improvement to the row andcolumn decoder approach employed in various prior art ISFET arraydesigns, including the design of Milgrew et al. shown in FIG. 7. Inparticular, regarding the row decoder 92 and the column decoder 94 shownin FIG. 7, the complexity of implementing these components in anintegrated circuit array design increases dramatically as the size ofthe array is increased, as additional inputs to both decoders arerequired. For example, an array having 512 rows and columns as discussedabove in connection with FIG. 13 would require nine inputs (2⁹=512) perrow and column decoder if such a scheme were employed for row and columnselection; similarly, arrays having 7400 rows and 7400 columns, asdiscussed below in connection with other embodiments, would require 13inputs (2¹³=8192) per row and column decoder. In contrast, the row andcolumn select shift registers shown in FIGS. 14 and 15 require noadditional input signals as array size is increased, but ratheradditional D-type flip-flops (which are routinely implemented in a CMOSprocess). Thus, the shift register implementations shown in FIGS. 14 and15 provide an easily scalable solution to array row and columnselection.

In the embodiment of FIG. 13, the “odd” column select shift register 194₁ provides odd column select signals to an “odd” output driver 198 ₁ andthe even column select shift register 194 ₂ provides even column selectsignals to an “even” output driver 198 ₂. Both output drivers areconfigured similarly, and an example of the even output driver 198 ₂ isshown in FIG. 16. In particular, FIG. 16 shows that respective evencolumn output signals V_(COL2), V_(COL4), . . . V_(COL512) (refer toFIG. 9 for the generic column signal output V_(COLj)) are applied tocorresponding switches 191 ₂, 191 ₄, . . . 191 ₅₁₂, responsive to theeven column select signals ColSel₂, ColSel₄, . . . ColSel₅₁₂ provided bythe column select register 194 ₂, to successively couple the even columnoutput signals to the input of a buffer amplifier 199 (BUF) via a bus175. In FIG. 16, the buffer amplifier 199 receives power from an outputbuffer positive supply voltage VDDO and an output buffer supply groundVSSO, and is responsive to an output buffer bias voltage VBO0 to set acorresponding bias current for the buffer output. Given the highimpedance input of the buffer amplifier 199, a current sink 197responsive to a bias voltage VB3 is coupled to the bus 175 to provide anappropriate drive current (e.g., on the order of approximately 100 μA)for the output of the column output buffer (see the buffer amplifier 111j of FIG. 9) of a selected column. The buffer amplifier 199 provides theoutput signal Vout2 based on the selected even column of the array; atthe same time, with reference to FIG. 13, a corresponding bufferamplifier of the “odd” output driver 198 ₁ provides the output signalVout1 based on a selected odd column of the array.

In one exemplary implementation, the switches of both the even and oddoutput drivers 198 ₁ and 198 ₂ (e.g., the switches 191 ₂, 191 ₄, . . .191 ₅₁₂ shown in FIG. 16) may be implemented as CMOS-pair transmissiongates (including an n-channel MOSFET and a p-channel MOSFET; see FIG.4), and inverters may be employed so that each column select signal andits complement may be applied to a given transmission gate switch 191 toenable switching. Each switch 191 has a series resistance when enabledor “on” to couple a corresponding column output signal to the bus 175;likewise, each switch adds a capacitance to the bus 175 when the switchis off A larger switch reduces series resistance and allows a higherdrive current for the bus 175, which generally allows the bus 175 tosettle more quickly; on the other hand, a larger switch increasescapacitance of the bus 175 when the switch is off, which in turnincreases the settling time of the bus 175. Hence, there is a trade-offbetween switch series resistance and capacitance in connection withswitch size.

The ability of the bus 175 to settle quickly following enabling ofsuccessive switches in turn facilitates rapid data acquisition from thearray. To this end, in some embodiments the switches 191 of the outputdrivers 198 ₁ and 198 ₂ are particularly configured to significantlyreduce the settling time of the bus 175. Both the n-channel and thep-channel MOSFETs of a given switch add to the capacitance of the bus175; however, n-channel MOSFETs generally have better frequency responseand current drive capabilities than their p-channel counterparts. Inview of the foregoing, Applicants have recognized and appreciated thatsome of the superior characteristics of n-channel MOSFETs may beexploited to improve settling time of the bus 175 by implementing“asymmetric” switches in which respective sizes for the n-channel MOSFETand p-channel MOSFET of a given switch are different.

For example, in one embodiment, with reference to FIG. 16, the currentsink 197 may be configured such that the bus 175 is normally “pulleddown” when all switches 191 ₂, 191 ₄, . . . 191 ₅₁₂ are open or off (notconducting). Given a somewhat limited expected signal dynamic range forthe column output signals based on ISFET measurements, when a givenswitch is enabled or on (conducting), in many instances most of theconduction is done by the n-channel MOSFET of the CMOS-pair constitutingthe switch. Accordingly, in one aspect of this embodiment, the n-channelMOSFET and the p-channel MOSFET of each switch 191 are sizeddifferently; namely, in one exemplary implementation, the n-channelMOSFET is sized to be significantly larger than the p-channel MOSFET.More specifically, considering equally-sized n-channel and p-channelMOSFETs as a point of reference, in one implementation the n-channelMOSFET may be increased to be about 2 to 2.5 times larger, and thep-channel MOSFET may be decreased in size to be about 8 to 10 timessmaller, such that the n-channel MOSFET is approximately 20 times largerthan the p-channel MOSFET. Due to the significant decrease in size ofthe p-channel MOSFET and the relatively modest increase in size of then-channel MOSFET, the overall capacitance of the switch in the off stateis notably reduced, and there is a corresponding notable reduction incapacitance for the bus 175; at the same time, due to the largern-channel MOSFET, there is a significant increase in current drivecapability, frequency response and transconductance of the switch, whichin turn results in a significant reduction in settling time of the bus175.

While the example above describes asymmetric switches 191 for the outputdrivers 198 ₁ and 198 ₂ in which the n-channel MOSFET is larger than thep-channel MOSFET, it should be appreciated that in another embodiment,the converse may be implemented, namely, asymmetric switches in whichthe p-channel MOSFET is larger than the n-channel MOSFET. In one aspectof this embodiment, with reference again to FIG. 16, the current sink197 may alternatively serve as a source of current to appropriatelydrive the output of the column output buffer (see the buffer amplifier111 j of FIG. 9) of a selected column, and be configured such that thebus 175 is normally “pulled up” when all switches 191 ₂, 191 ₄, . . .191 ₅₁₂ are open or off (not conducting). In this situation, most of theswitch conduction may be accomplished by the p-channel MOSFET of theCMOS-pair constituting the switch. Benefits of reduced switchcapacitance (and hence reduced bus capacitance) may be realized in thisembodiment, although the overall beneficial effect of reduced settlingtime for the bus 175 may be somewhat less than that described previouslyabove, due to the lower frequency response of p-channel MOSFETs ascompared to n-channel MOSFETs. Nevertheless, asymmetric switches basedon larger p-channel MOSFETs may still facilitate a notable reduction inbus settling time, and may also provide for circuit implementations inwhich the column output buffer amplifier (111 j of FIG. 9) may be abody-tied source follower with appreciably increased gain.

In yet another embodiment directed to facilitating rapid settling of thebus 175 shown in FIG. 16, it may be appreciated that fewer switches 191coupled to the bus 175 results in a smaller bus capacitance. With thisin mind, and with reference again to FIG. 13, in yet another embodiment,more than two output drivers 198 ₁ and 198 ₂ may be employed in theISFET array 100 such that each output driver handles a smaller number ofcolumns of the array. For example, rather than having all even columnshandled by one driver and all odd columns handled by another driver, thearray may include four column select registers 194 _(1,2,3,4) and fourcorresponding output drivers 198 _(1,2,3,4) such that each output driverhandles one-fourth of the total columns of the array, rather thanone-half of the columns. In such an implementation, each output driverwould accordingly have half the number of switches 191 as compared withthe embodiment discussed above in connection with FIG. 16, and the bus175 of each output driver would have a corresponding lower capacitance,thereby improving bus settling time. While four output drivers arediscussed for purposes of illustration in this example, it should beappreciated that the present disclosure is not limited in this respect,and virtually any number of output drivers greater than two may beemployed to improve bus settling time in the scenario described above.Other array embodiments in which more than two output drivers areemployed to facilitate rapid data acquisition from the array arediscussed in greater detail below (e.g., in connection with FIGS.19-23).

For purposes of illustration, the bus 175 may have a capacitance in therange of approximately 5 pF to 20 pF in any of the embodiments discussedimmediately above (e.g. symmetric switches, asymmetric switches, greaternumbers of output drivers, etc.). Of course, it should be appreciatedthat the capacitance of the bus 175 is not limited to these exemplaryvalues, and that other capacitance values are possible in differentimplementations of an array according to the present disclosure.

In one aspect of the array design discussed above in connection withFIGS. 13-16, separate analog supply voltage connections (for VDDA,VSSA), digital supply voltage connections (for VDDD, VSSD) and outputbuffer supply voltage connections (for VDDO, VSSO) are provided on thearray to facilitate noise isolation and reduce signal cross-talk amongstvarious array components, thereby increasing the signal-to-noise ratio(SNR) of the output signals Vout1 and Vout2. In one exemplaryimplementation, the positive supply voltages VDDA, VDDD and VDDO eachmay be approximately 3.3 Volts. In another aspect, these voltagesrespectively may be provided “off chip” by one or more programmablevoltage sources, as discussed further below in connection with FIG. 17.

FIG. 17 illustrates a block diagram of the sensor array 100 of FIG. 13coupled to an array controller 250, according to one inventiveembodiment of the present disclosure. In various exemplaryimplementations, the array controller 250 may be fabricated as a “standalone” controller, or as one or more computer compatible “cards” formingpart of a computer 260, as discussed above in connection with FIG. 8. Inone aspect, the functions of the array controller 250 may be controlledby the computer 260 through an interface block 252 (e.g., serialinterface, via USB port or PCI bus, Ethernet connection, etc.), as shownin FIG. 17. In one embodiment, all or a portion of the array controller250 is fabricated as one or more printed circuit boards, and the array100 is configured to plug into one of the printed circuit boards,similar to a conventional IC chip (e.g., the array 100 is configured asan ASIC that plugs into a chip socket, such as a zero-insertion-force or“ZIF” socket, of a printed circuit board). In one aspect of such anembodiment, an array 100 configured as an ASIC may include one or morepins/terminal connections dedicated to providing an identification code,indicated as “ID” in FIG. 17, that may be accessed/read by the arraycontroller 250 and/or passed on to the computer 260. Such anidentification code may represent various attributes of the array 100(e.g., size, number of pixels, number of output signals, variousoperating parameters such as supply and/or bias voltages, etc.) and maybe processed to determine corresponding operating modes, parameters andor signals provided by the array controller 250 to ensure appropriateoperation with any of a number of different types of arrays 100. In oneexemplary implementation, an array 100 configured as an ASIC may beprovided with three pins dedicated to an identification code, and duringthe manufacturing process the ASIC may be encoded to provide one ofthree possible voltage states at each of these three pins (i.e., atri-state pin coding scheme) to be read by the array controller 250,thereby providing for 27 unique array identification codes. In anotheraspect of this embodiment, all or portions of the array controller 250may be implemented as a field programmable gate array (FPGA) configuredto perform various array controller functions described in furtherdetail below.

Generally, the array controller 250 provides various supply voltages andbias voltages to the array 100, as well as various signals relating torow and column selection, sampling of pixel outputs and dataacquisition. In particular, the array controller 250 reads one or moreanalog output signals (e.g., Vout1 and Vout2) including multiplexedrespective pixel voltage signals from the array 100 and then digitizesthese respective pixel signals to provide measurement data to thecomputer 260, which in turn may store and/or process the data. In someimplementations, the array controller 250 also may be configured toperform or facilitate various array calibration and diagnosticfunctions, and an optional array UV irradiation treatment as discussedabove in connection with FIG. 11A.

As illustrated in FIG. 17, the array controller 250 generally providesto the array 100 the analog supply voltage and ground (VDDA, VSSA), thedigital supply voltage and ground (VDDD, VSSD), and the buffer outputsupply voltage and ground (VDDO, VSSO). In one exemplary implementation,each of the supply voltages VDDA, VDDD and VDDO is approximately 3.3Volts. In another implementation, the supply voltages VDDA, VDDD andVDDO may be as low as approximately 1.8 Volts. As discussed above, inone aspect each of these power supply voltages is provided to the array100 via separate conducting paths to facilitate noise isolation. Inanother aspect, these supply voltages may originate from respectivepower supplies/regulators, or one or more of these supply voltages mayoriginate from a common source in a power supply 258 of the arraycontroller 250. The power supply 258 also may provide the various biasvoltages required for array operation (e.g., VB1, VB2, VB3, VB4, VBO0,V_(BODY)) and the reference voltage VREF used for array diagnostics andcalibration.

In another aspect, the power supply 258 includes one or moredigital-to-analog converters (DACs) that may be controlled by thecomputer 260 to allow any or all of the bias voltages, referencevoltage, and supply voltages to be changed under software control (i.e.,programmable bias settings). For example, a power supply 258 responsiveto computer control (e.g., via software execution) may facilitateadjustment of one or more of the supply voltages (e.g., switchingbetween 3.3 Volts and 1.8 Volts depending on chip type as represented byan identification code), and or adjustment of one or more of the biasvoltages VB1 and VB2 for pixel drain current, VB3 for column bus drive,VB4 for column amplifier bandwidth, and VBO0 for column output buffercurrent drive. In some aspects, one or more bias voltages may beadjusted to optimize settling times of signals from enabled pixels.Additionally, the common body voltage V_(BODY) for all ISFETs of thearray may be grounded during an optional post-fabrication UV irradiationtreatment to reduce trapped charge, and then coupled to a higher voltage(e.g., VDDA) during diagnostic analysis, calibration, and normaloperation of the array for measurement/data acquisition. Likewise, thereference voltage VREF may be varied to facilitate a variety ofdiagnostic and calibration functions.

As also shown in FIG. 17, the reference electrode 76 which is typicallyemployed in connection with an analyte solution to be measured by thearray 100 (as discussed above in connection with FIG. 1), may be coupledto the power supply 258 to provide a reference potential for the pixeloutput voltages. For example, in one implementation the referenceelectrode 76 may be coupled to a supply ground (e.g., the analog groundVSSA) to provide a reference for the pixel output voltages based on Eq.(3) above. In other exemplary implementations, the reference electrodevoltage may be set by placing a solution/sample of interest having aknown pH level in proximity to the sensor array 100 and adjusting thereference electrode voltage until the array output signals Vout1 andVout2 provide pixel voltages at a desired reference level, from whichsubsequent changes in pixel voltages reflect local changes in pH withrespect to the known reference pH level. In general, it should beappreciated that a voltage associated with the reference electrode 76need not necessarily be identical to the reference voltage VREFdiscussed above (which may be employed for a variety of array diagnosticand calibration functions), although in some implementations thereference voltage VREF provided by the power supply 258 may be used toset the voltage of the reference electrode 76.

Regarding data acquisition from the array 100, in one embodiment thearray controller 250 of FIG. 17 may include one or more preamplifiers253 to further buffer one or more output signals (e.g., Vout1 and Vout2)from the sensor array and provide selectable gain. In one aspect, thearray controller 250 may include one preamplifier for each output signal(e.g., two preamplifiers for two analog output signals). In otheraspects, the preamplifiers may be configured to accept input voltagesfrom 0.0 to 1.8 Volts or 0.0 to 3.3 Volts, may haveprogrammable/computer selectable gains (e.g., 1, 2, 5, 10 and 20) andlow noise outputs (e.g., <10 nV/sqrtHz), and may provide low passfiltering (e.g., bandwidths of 5 MHz and 25 MHz). With respect to noisereduction and increasing signal-to-noise ratio, in one implementation inwhich the array 100 is configured as an ASIC placed in a chip socket ofa printed circuit board containing all or a portion of the arraycontroller 250, filtering capacitors may be employed in proximity to thechip socket (e.g., the underside of a ZIF socket) to facilitate noisereduction. In yet another aspect, the preamplifiers may have aprogrammable/computer selectable offset for input and/or output voltagesignals to set a nominal level for either to a desired range.

The array controller 250 of FIG. 17 also comprises one or moreanalog-to-digital converters 254 (ADCs) to convert the sensor arrayoutput signals Vout1 and Vout2 to digital outputs (e.g., 10-bit or12-bit) so as to provide data to the computer 260. In one aspect, oneADC may be employed for each analog output of the sensor array, and eachADC may be coupled to the output of a corresponding preamplifier (ifpreamplifiers are employed in a given implementation). In anotheraspect, the ADC(s) may have a computer-selectable input range (e.g., 50mV, 200 mV, 500 mV, 1V) to facilitate compatibility with differentranges of array output signals and/or preamplifier parameters. In yetother aspects, the bandwidth of the ADC(s) may be greater than 60 MHz,and the data acquisition/conversion rate greater than 25 MHz (e.g., ashigh as 100 MHz or greater).

In the embodiment of FIG. 17, ADC acquisition timing and array row andcolumn selection may be controlled by a timing generator 256. Inparticular, the timing generator provides the digital vertical data andclock signals (DV, CV) to control row selection, the digital horizontaldata and clock signals (DH, CH) to control column selection, and thecolumn sample and hold signal COL SH to sample respective pixel voltagesfor an enabled row, as discussed above in connection with FIG. 9. Thetiming generator 256 also provides a sampling clock signal CS to theADC(s) 254 so as to appropriately sample and digitize consecutive pixelvalues in the data stream of a given array analog output signal (e.g.,Vout1 and Vout2), as discussed further below in connection with FIG. 18.In some implementations, the timing generator 256 may be implemented bya microprocessor executing code and configured as a multi-channeldigital pattern generator to provide appropriately timed controlsignals. In one exemplary implementation, the timing generator 256 maybe implemented as a field-programmable gate array (FPGA).

FIG. 18 illustrates an exemplary timing diagram for various arraycontrol signals, as provided by the timing generator 256, to acquirepixel data from the sensor array 100. For purposes of the followingdiscussion, a “frame” is defined as a data set that includes a value(e.g., pixel output signal or voltage V_(S)) for each pixel in thearray, and a “frame rate” is defined as the rate at which successiveframes may be acquired from the array. Thus, the frame rate correspondsessentially to a “pixel sampling rate” for each pixel of the array, asdata from any given pixel is obtained at the frame rate.

In the example of FIG. 18, an exemplary frame rate of 20 frames/sec ischosen to illustrate operation of the array (i.e., row and columnselection and signal acquisition); however, it should be appreciatedthat arrays and array controllers according to the present disclosureare not limited in this respect, as different frame rates, includinglower frame rates (e.g., 1 to 10 frames/second) or higher frame rates(e.g., 25, 30, 40, 50, 60, 70 to 100 frames/sec., etc.), with arrayshaving the same or higher numbers of pixels, are possible. In someexemplary applications, a data set may be acquired that includes manyframes over several seconds to conduct an experiment on a given analyteor analytes. Several such experiments may be performed in succession, insome cases with pauses in between to allow for data transfer/processingand/or washing of the sensor array ASIC and reagent preparation for asubsequent experiment.

For example, with respect to the method for detecting nucleotideincorporation, discussed above in connection with FIGS. 8, 8A and 8B, inwhich one or more ion pulses are generated in the output signal of agiven ISFET pixel of the array during a nucleic acid synthesis orsequencing reaction in a reaction well above the ISFET, appropriateframe rates may be chosen to sufficiently sample the ISFET's outputsignal so as to effectively detect the presence of one or more pulsesand the time interval between pulses. In some exemplary implementations,one or more ion pulses may be generated having a full-width athalf-maximum (FWHM) on the order of approximately 1 second toapproximately 2.5 seconds, and time intervals between successive pulsepeaks (if multiple pulses are generated) on the order of approximately 1to 20 seconds, depending on the number of nucleotide incorporationevents. Given these exemplary values, a frame rate (or pixel samplingrate) of 20 Hz is sufficient to reliably resolve the one or more pulsesin a given pixel's output signal. Again, the pulse characteristics andframe rate given in this example are provided primarily for purposes ofillustration, and different pulse characteristics and frame rates may beinvolved in other implementations.

In one implementation, the array controller 250 controls the array 100to enable rows successively, one at a time. For example, with referenceagain for the moment to FIG. 9, a first row of pixels is enabled via therow select signal RowSel₁ . The enabled pixels are allowed to settle forsome time period, after which the COL SH signal is asserted briefly toclose the sample/hold switch in each column and store on the column'ssample/hold capacitor C_(sh) the voltage value output by the first pixelin the column. This voltage is then available as the column outputvoltage V_(COLj) applied to one of the two (odd and even column) arrayoutput drivers 198 ₁ and 198 ₂ (e.g., see FIG. 16). The COL SH signal isthen de-asserted, thereby opening the sample/hold switches in eachcolumn and decoupling the column output buffer 111 j from the columnamplifiers 107A and 107B. Shortly thereafter, the second row of pixelsis enabled via the row select signal RowSel₂ . During the time period inwhich the second row of pixels is allowed to settle, the column selectsignals are generated two at a time (one odd and one even; odd columnselect signals are applied in succession to the odd output driver, evencolumn select signals are applied in succession to the even outputdriver) to read the column output voltages associated with the firstrow. Thus, while a given row in the array is enabled and settling, theprevious row is being read out, two columns at a time. By staggering rowselection and sampling/readout (e.g., via different vertical andhorizontal clock signals and column sample/hold), and by readingmultiple columns at a time for a given row, a frame of data may beacquired from the array in a significantly streamlined manner.

FIG. 18 illustrates the timing details of the foregoing process for anexemplary frame rate of 20 frames/sec. Given this frame rate and 512rows in the array, each row must be read out in approximately 98microseconds, as indicated by the vertical delineations in FIG. 18.Accordingly, the vertical clock signal CV has a period of 98microseconds (i.e., a clock frequency of over 10 kHz), with a new rowbeing enabled on a trailing edge (negative transition) of the CV signal.The left side of FIG. 18 reflects the beginning of a new frame cycle, atwhich point the vertical data signal DV is asserted before a firsttrailing edge of the CV signal and de-asserted before the next trailingedge of the CV signal (for data acquisition from successive frames, thevertical data signal is reasserted again only after row 512 is enabled).Also, immediately before each trailing edge of the CV signal (i.e., newrow enabled), the COL SH signal is asserted for 2 microseconds, leavingapproximately 50 nanoseconds before the trailing edge of the CV signal.

In FIG. 18, the first occurrence of the COL SH signal is actuallysampling the pixel values of row 512 of the array. Thus, upon the firsttrailing edge of the CV signal, the first row is enabled and allowed tosettle (for approximately 96 microseconds) until the second occurrenceof the COL SH signal. During this settling time for the first row, thepixel values of row 512 are read out via the column select signals.Because two column select signals are generated simultaneously to read512 columns, the horizontal clock signal CH must generate 256 cycleswithin this period, each trailing edge of the CH signal generating oneodd and one even column select signal. As shown in FIG. 18, the firsttrailing edge of the CH signal in a given row is timed to occur twomicroseconds after the selection of the row (after deactivation of theCOL SH signal) to allow for settling of the voltage values stored on thesample/hold capacitors C_(sh) and provided by the column output buffers.It should be appreciated however that, in other implementations (e.g.,as discussed below in connection with FIG. 18A), the time period betweenthe first trailing edge of the CH signal and a trailing edge (i.e.,deactivation) of the COL SH signal may be significantly less than twomicroseconds, and in some cases as small as just over 50 nanoseconds.Also for each row, the horizontal data signal DH is asserted before thefirst trailing edge of the CH signal and de-asserted before the nexttrailing edge of the CH signal. The last two columns (e.g., 511 and 512)are selected before the occurrence of the COL SH signal which, asdiscussed above, occurs approximately two microseconds before the nextrow is enabled. Thus, 512 columns are read, two at a time, within a timeperiod of approximately 94 microseconds (i.e., 98 microseconds per row,minus two microseconds at the beginning and end of each row). Thisresults in a data rate for each of the array output signals Vout1 andVout2 of approximately 2.7 MHz.

FIG. 18A illustrates another timing diagram of a data acquisitionprocess from an array 100 that is slightly modified from the timingdiagram of FIG. 18. As discussed above in connection with FIG. 13, insome implementations an array similar to that shown in FIG. 13 may beconfigured to include a region of 512 by 512 “active” pixels that aresurrounded by a perimeter of reference pixels (i.e., the first and lasttwo rows and columns of the array), resulting in an array having a totalpixel count of 516 by 516 pixels. Accordingly, given the exemplary framerate of 20 frames/sec and 516 rows in the array, each row must be readout in approximately 97 microseconds, as indicated by the verticaldelineations in FIG. 18A. Accordingly, the vertical clock signal CV hasa slightly smaller period of 97 microseconds. Because two column selectsignals are generated simultaneously to read 516 columns, the horizontalclock signal CH must generate 258 cycles within this period, as opposedto the 256 cycles referenced in connection with FIG. 18. Accordingly, inone aspect illustrated in FIG. 18A, the first trailing edge of the CHsignal in a given row is timed to occur just over 50 nanoseconds fromthe trailing edge (i.e., deactivation) of the COL SH signal, so as to“squeeze” additional horizontal clock cycles into a slightly smallerperiod of the vertical clock signal CV. As in FIG. 18, the horizontaldata signal DH is asserted before the first trailing edge of the CHsignal, and as such also occurs slightly earlier in the timing diagramof FIG. 18A as compared to FIG. 18. The last two columns (i.e., columns515 and 516, labeled as “Ref3,4 in FIG. 18A) are selected before theoccurrence of the COL SH signal which, as discussed above, occursapproximately two microseconds before the next row is enabled. Thus, 516columns are read, two at a time, within a time period of approximately95 microseconds (i.e., 97 microseconds per row, minus two microsecondsat the end of each row and negligible time at the beginning of eachrow). This results in essentially the same data rate for each of thearray output signals Vout1 and Vout2 provided by the timing diagram ofFIG. 18, namely, approximately 2.7 MHz.

As discussed above in connection with FIG. 17, the timing generator 256also generates the sampling clock signal CS to the ADC(s) 254 so as toappropriately sample and digitize consecutive pixel values in the datastream of a given array output signal. In one aspect, the sampling clocksignal CS provides for sampling a given pixel value in the data streamat least once. Although the sampling clock signal CS is not shown in thetiming diagrams of FIGS. 18 and 18A, it may be appreciated that inexemplary implementations the signal CS may essentially track the timingof the horizontal clock signal CH; in particular, the sampling clocksignal CS may be coordinated with the horizontal clock signal CH so asto cause the ADC(s) to sample a pixel value immediately prior to a nextpixel value in the data stream being enabled by CH, thereby allowing foras much signal settling time as possible prior to sampling a given pixelvalue. For example, the ADC(s) may be configured to sample an inputpixel value upon a positive transition of CS, and respective positivetransitions of CS may be timed by the timing generator 256 to occurimmediately prior to, or in some cases essentially coincident with,respective negative transitions of CH, so as to sample a given pixeljust prior to the next pixel in the data stream being enabled. Inanother exemplary implementation, the ADC(s) 254 may be controlled bythe timing generator 256 via the sampling clock signal CS to sample theoutput signals Vout1 and Vout2 at a significantly higher rate to providemultiple digitized samples for each pixel measurement, which may then beaveraged (e.g., the ADC data acquisition rate may be approximately 100MHz to sample the 2.7 MHz array output signals, thereby providing asmany as approximately 35-40 samples per pixel measurement).

In one embodiment, once pixel values are sampled and digitized by theADC(s) 254, the computer 260 may be programmed to process pixel dataobtained from the array 100 and the array controller 250 so as tofacilitate high data acquisition rates that in some cases may exceed asufficient settling time for pixel voltages represented in a given arrayoutput signal. A flow chart illustrating an exemplary method accordingto one embodiment of the present invention that may be implemented bythe computer 260 for processing and correction of array data acquired athigh acquisition rates is illustrated in FIG. 18B. In various aspects ofthis embodiment, the computer 260 is programmed to first characterize asufficient settling time for pixel voltages in a given array outputsignal, as well as array response at appreciably high operatingfrequencies, using a reference or “dry” input to the array (e.g., noanalyte present). This characterization forms the basis for derivingcorrection factors that are subsequently applied to data obtained fromthe array at the high operating frequencies and in the presence of ananalyte to be measured.

Regarding pixel settling time, with reference again to FIG. 16, asdiscussed above a given array output signal (e.g., Vout2 in FIG. 16)includes a series of pixel voltage values resulting from the sequentialoperation of the column select switches 191 to apply respective columnvoltages V_(COLj) via the bus 175 to the buffer amplifier 199 (therespective column voltages V_(COLj) in turn represent buffered versionsof ISFET source voltages V_(Sj)). In some implementations, it isobserved that voltage changes ΔV_(PIX) in the array output signalbetween two consecutive pixel reads is characterized as an exponentialprocess given byΔV _(PIX)(t)=A(1−e ^(−t/k)),  (PP)where A is the difference (V_(COLj)−V_(COLj−1)) between two pixelvoltage values and k is a time constant associated with a capacitance ofthe bus 175. FIGS. 18C and 18D illustrate exemplary pixel voltages in agiven array output signal Vout (e.g., one of Vout1 and Vout2) showingpixel-to-pixel transitions in the output signal as a function of time,plotted against exemplary sampling clock signals CS. In FIG. 18C, thesampling clock signal CS has a period 524, and an ADC controlled by CSsamples a pixel voltage upon a positive transition of CS (as discussedabove, in one implementation CS and CH have essentially a same period).FIG. 18C indicates two samples 525A and 525B, between which anexponential voltage transition 522 corresponding to ΔV_(PIX)(t), betweena voltage difference A, may be readily observed.

For purposes of the present discussion, pixel “settling time” t_(settle)is defined as the time t at which ΔV_(PIX)(t) attains a value thatdiffers from it's final value by an amount that is equal to the peaknoise level of the array output signal. If the peak noise level of thearray output signal is denoted as n_(p), then the voltage at thesettling time t_(settle) is given byΔV_(PIX)(t_(settle))=A[1−(n_(p)/A)]. Substituting in Eq. (PP) andsolving for t_(settle) yields

$\begin{matrix}{t_{settle} = {{- k}\;{{\ln\left( \frac{n_{p}}{A} \right)}.}}} & ({QQ})\end{matrix}$FIG. 18D conceptually illustrates a pixel settling time t_(settle)(reference numeral 526) for a single voltage transition 522 between twopixel voltages having a difference A, using a sampling clock signal CShaving a sufficiently long period so as to allow for full settling. Toprovide some exemplary parameters for purposes of illustration, in oneimplementation a maximum value for A, representing a maximum range forpixel voltage transitions (e.g., consecutive pixels at minimum andmaximum values), is on the order of approximately 250 mV. Additionally,a peak noise level n_(p) of the array output signal is taken asapproximately 100 μV, and the time constant k is taken as 5 nanoseconds.These values provide an exemplary settling time t_(settle) ofapproximately 40 nanoseconds. If a maximum data rate of an array outputsignal is taken as the inverse of the settling time t_(settle), asettling time of 40 nanoseconds corresponds to maximum data rate of 25MHz. In other implementations, A may be on the order of 20 mV and thetime constant k may be on the order of 15 nanoseconds, resulting in asettling time t_(settle) of approximately 80 nanoseconds and a maximumdata rate of 12.5 MHz. The values of k indicated above generallycorrespond to capacitances for the bus 175 in a range of approximately 5pF to 20 pF. It should be appreciated that the foregoing values areprovided primarily for purposes of illustration, and that variousembodiments of the present invention are not limited to these exemplaryvalues; in particular, arrays according to various embodiment of thepresent invention may have different pixel settling times t_(settle)(e.g., in some cases less than 40 nanoseconds).

As indicated above, in one embodiment pixel data may be acquired fromthe array at data rates that exceed those dictated by the pixel settlingtime. FIG. 18B illustrates a flow chart for such a method according toone inventive embodiment of the present disclosure. In the method ofFIG. 18B, sufficiently slow clock frequencies initially are chosen forthe signals CV, CH and CS such that the resulting data rate per arrayoutput signal is equal to or lower than the reciprocal of the pixelsettling time t_(settle) to allow for fully settled pixel voltage valuesfrom pixel to pixel in a given output signal. With these clockfrequencies, as indicated in block 502 of FIG. 18B, settled pixelvoltage values are then acquired for the entire array in the absence ofan analyte (or in the presence of a reference analyte) to provide afirst “dry” or reference data image for the array. In block 504 of FIG.18B, for each pixel voltage constituting the first data image, atransition value between the pixel's final voltage and the final voltageof the immediately preceding pixel in the corresponding output signaldata stream (i.e., the voltage difference A) is collected and stored.The collection of these transition values for all pixels of the arrayprovides a first transition value data set.

Subsequently, in block 506 of FIG. 18B, the clock frequencies for thesignals CV, CH and CS are increased such that the resulting data rateper array output signal exceeds a rate at which pixel voltage values arefully settled (i.e., a data rate higher than the reciprocal of thesettling time t_(settle)). For purposes of the present discussion, thedata rate per array output signal resulting from the selection of suchincreased clock frequencies for the signals CV, CH and CS is referred toas an “overspeed data rate.” Using the clock frequencies correspondingto the overspeed data rate, pixel voltage values are again obtained forthe entire array in the absence of an analyte (or in the presence of thesame reference analyte) to provide a second “dry” or reference dataimage for the array. In block 508 of FIG. 18B, a second transition valuedata set based on the second data image obtained at the overspeed datarate is calculated and stored, as described above for the first dataimage.

In block 510 of FIG. 18B, a correction factor for each pixel of thearray is calculated based on the values stored in the first and secondtransition value data sets. For example, a correction factor for eachpixel may be calculated as a ratio of its transition value from thefirst transition value data set and its corresponding transition valuefrom the second transition value data set (e.g., the transition valuefrom the first data set may be divided by the transition value from thesecond data set, or vice versa) to provide a correction factor data setwhich is then stored. As noted in blocks 512 and 514 of FIG. 18B, thiscorrection factor data set may then be employed to process pixel dataobtained from the array operated at clock frequencies corresponding tothe overspeed data rate, in the presence of an actual analyte to bemeasured; in particular, data obtained from the array at the overspeeddata rate in the presence of an analyte may be multiplied or divided asappropriate by the correction factor data set (e.g., each pixelmultiplied or divided by a corresponding correction factor) to obtaincorrected data representative of the desired analyte property to bemeasured (e.g., ion concentration). It should be appreciated that oncethe correction factor data set is calculated and stored, it may be usedrepeatedly to correct multiple frames of data acquired from the array atthe overspeed data rate.

In addition to controlling the sensor array and ADCs, the timinggenerator 256 may be configured to facilitate various array calibrationand diagnostic functions, as well as an optional UV irradiationtreatment. To this end, the timing generator may utilize the signal LSTVindicating the selection of the last row of the array and the signalLSTH to indicate the selection of the last column of the array. Thetiming generator 256 also may be responsible for generating the CALsignal which applies the reference voltage VREF to the column bufferamplifiers, and generating the UV signal which grounds the drains of allISFETs in the array during a UV irradiation process (see FIG. 9). Thetiming generator also may provide some control function over the powersupply 258 during various calibration and diagnostic functions, or UVirradiation, to appropriately control supply or bias voltages; forexample, during UV irradiation, the timing generator may control thepower supply to couple the body voltage V_(BODY) to ground while the UVsignal is activated to ground the ISFET drains. With respect to arraycalibration and diagnostics, as well as UV irradiation, in someimplementations the timing generator may receive specialized programsfrom the computer 260 to provide appropriate control signals. In oneaspect, the computer 260 may use various data obtained from referenceand/or dummy pixels of the array, as well as column information based onthe application of the CAL signal and the reference voltage VREF, todetermine various calibration parameters associated with a given arrayand/or generate specialized programs for calibration and diagnosticfunctions.

With respect to the computer interface 252 of the array controller 250,in one exemplary implementation the interface is configured tofacilitate a data rate of approximately 200 MB/sec to the computer 260,and may include local storage of up to 400 MB or greater. The computer260 is configured to accept data at a rate of 200 MB/sec, and processthe data so as to reconstruct an image of the pixels (e.g., which may bedisplayed in false-color on a monitor). For example, the computer may beconfigured to execute a general-purpose program with routines written inC++ or Visual Basic to manipulate the data and display is as desired.

The systems described herein, when used for sequencing, typicallyinvolve a chemFET array supporting reaction chambers, the chemFETs beingcoupled to an interface capable of executing logic that converts thesignals from the chemFETs into sequencing information.

In some embodiments, the invention encompasses logic (preferablycomputer executable logic) for polymer sequencing, comprising logic fordetermining ion pulses associated with an ionic interaction with a PPior a dNTP or both. Typically, the logic converts characteristic(s) ofthe ion pulses into polymer sequencing information.

In some embodiments, the invention encompasses logic (preferablycomputer executable logic) comprising logic for determining a sequenceof a nucleic acid template based on time between ion pulses or acharacteristic of a single ion pulse. The logic may optionally furthercomprise logic for determining spatial location of the ion pulse on anarray of chemFETs.

In some embodiments, the invention encompasses logic (preferablycomputer executable logic) comprising logic for determining a sequenceof a nucleic acid template based on a duration of time it takes for aparticular dNTP to be utilized in a sequencing reaction. Typically, thelogic receives signal from one or more chemFETs. Preferably, thesequence is displayed in substantially real time.

In some embodiments, the invention encompasses logic (preferablycomputer executable logic) for processing ion pulses from an array ofchemFETs to determine the sequence of a polymer of interest. The logicmay optionally further comprise logic for file management, file storage,and visualization. The logic may also optionally further comprise logicfor converting the ion pulses into nucleotide sequences. Preferably, thesequence is displayed in substantially real time.

The sequencing information obtained from the system may be delivered toa handheld computing device, such as a personal digital assistant. Thus,in one embodiment, the invention encompasses logic for displaying acomplete genome of an organism on a handheld computing device. Theinvention also encompasses logic adapted for sending data from a chemFETarray to a handheld computing device. Any of such logic may becomputer-implemented.

Having discussed several aspects of an exemplary ISFET array and anarray controller according to the present disclosure, FIGS. 19-23illustrate block diagrams of alternative CMOS IC chip implementations ofISFET sensor arrays having greater numbers of pixels, according to yetother inventive embodiments. In one aspect, each of the ISFET arraysdiscussed further below in connection with FIGS. 19-23 may be controlledby an array controller similar to that shown in FIG. 17, in some caseswith minor modifications to accommodate higher numbers of pixels (e.g.,additional preamplifiers 253 and analog-to-digital converters 254).

FIG. 19 illustrates a block diagram of an ISFET sensor array 100A basedon the column and pixel designs discussed above in connection with FIGS.9-12 and a 0.35 micrometer CMOS fabrication process, according to oneinventive embodiment. The array 100A includes 2048 columns 102 ₁ through102 ₂₀₄₈, wherein each column includes 2048 geometrically square pixels105 ₁ through 105 ₂₀₄₈, each having a size of approximately 9micrometers by 9 micrometers. Thus, the array includes over four millionpixels (>4 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 20.5millimeters by 20.5 millimeters.

In one aspect of the embodiment shown in FIG. 19, the array 100A may beconfigured, at least in part, as multiple groups of pixels that may berespectively controlled. For example, each column of pixels may bedivided into top and bottom halves, and the collection of pixels inrespective top halves of columns form a first group 400 ₁ of rows (e.g.,a top group, rows 1-1024) and the collection of pixels in respectivebottom halves of columns form a second group 400 ₂ of rows (e.g., abottom group, rows 1025-2048). In turn, each of the first and second(e.g., top and bottom) groups of rows is associated with correspondingrow select registers, column bias/readout circuitry, column selectregisters, and output drivers. In this manner, pixel selection and dataacquisition from each of the first and second groups of rows 400 ₁ and400 ₂ is substantially similar to pixel selection and data acquisitionfrom the entire array 100 shown in FIG. 13; stated differently, in oneaspect, the array 100A of FIG. 19 substantially comprises twosimultaneously controlled “sub-arrays” of different pixel groups toprovide for significantly streamlined data acquisition from highernumbers of pixels.

In particular, FIG. 19 shows that row selection of the first group 400 ₁of rows may be controlled by a first row select register 192 ₁, and rowselection of the second group 400 ₂ of rows may be controlled by asecond row select register 192 ₂. In one aspect, each of the row selectregisters 192 ₁ and 192 ₂ may be configured as discussed above inconnection with FIG. 14 to receive vertical clock (CV) and vertical data(DV) signals and generate row select signals in response; for examplethe first row select register 192 ₁ may generate the signals RowSel₁through RowSel₁₀₂₄ and the second row select register 192 ₂ may generatethe signals RowSel₁₀₂₅ through RowSel₂₀₄₈ . In another aspect, both rowselect registers 192 ₁ and 192 ₂ may simultaneously receive commonvertical clock and data signals, such that two rows of the array areenabled at any given time, one from the top group and another from thebottom group.

For each of the first and second groups of rows, the array 100A of FIG.19 further comprises column bias/readout circuitry 110 _(1T)-110_(2048T) (for the first row group 400 ₁) and 110 _(1B)-110 _(2048B) (forthe second row group 400 ₂), such that each column includes twoinstances of the bias/readout circuitry 110 j shown in FIG. 9. The array100A also comprises two column select registers 192 _(1,2) (odd andeven) and two output drivers 198 _(1,2) (odd and even) for the secondrow group 400 ₂, and two column select registers 192 _(3,4) (odd andeven) and two output drivers 198 _(3,4) (odd and even) for the first rowgroup 400 ₁ (i.e., a total of four column select registers and fouroutput drivers). The column select registers receive horizontal clocksignals (CHT and CHB for the first row group and second row group,respectively) and horizontal data signals (DHT and DHB for the first rowgroup and second row group, respectively) to control odd and even columnselection. In one implementation, the CHT and CHB signals may beprovided as common signals, and the DHT and DHB may be provided ascommon signals, to simultaneously read out four columns at a time fromthe array (i.e., one odd and one even column from each row group); inparticular, as discussed above in connection with FIGS. 13-18, twocolumns may be simultaneously read for each enabled row and thecorresponding pixel voltages provided as two output signals. Thus, viathe enablement of two rows at any given time, and reading of two columnsper row at any given time, the array 100A may provide four simultaneousoutput signals Vout1, Vout2, Vout3 and Vout4.

In one exemplary implementation of the array 100A of FIG. 19, in whichcomplete data frames (all pixels from both the first and second rowgroups 400 ₁ and 400 ₂) are acquired at a frame rate of 20 frames/sec,1024 pairs of rows are successively enabled for periods of approximately49 microseconds each. For each enabled row, 1024 pixels are read out byeach column select register/output driver during approximately 45microseconds (allowing 2 microseconds at the beginning and end of eachrow, as discussed above in connection with FIG. 18). Thus, in thisexample, each of the array output signals Vout1, Vout2, Vout3 and Vout4has a data rate of approximately 23 MHz. Again, it should be appreciatedthat in other implementations, data may be acquired from the array 100Aof FIG. 19 at frame rates other than 20 frames/sec (e.g., 50-100frames/sec).

Like the array 100 of FIG. 13, in yet other aspects the array 100A ofFIG. 19 may include multiple rows and columns of dummy or referencepixels 103 around a perimeter of the array to facilitate preliminarytest/evaluation data, offset determination an/or array calibration.Additionally, various power supply and bias voltages required for arrayoperation (as discussed above in connection with FIG. 9) are provided tothe array 100A in block 195, in a manner similar to that discussed abovein connection with FIG. 13.

FIG. 20 illustrates a block diagram of an ISFET sensor array 100B basedon a 0.35 micrometer CMOS fabrication process and having a configurationsubstantially similar to the array 100A discussed above in FIG. 19,according to yet another inventive embodiment. While the array 100B alsois based generally on the column and pixel designs discussed above inconnection with FIGS. 9-12, the pixel size/pitch in the array 100B issmaller than that of the pixel shown in FIG. 10. In particular, withreference again to FIGS. 10 and 11, the dimension “e” shown in FIG. 10is substantially reduced in the embodiment of FIG. 20, without affectingthe integrity of the active pixel components disposed in the centralregion of the pixel, from approximately 9 micrometers to approximately 5micrometers; similarly, the dimension “f” shown in FIG. 10 is reducedfrom approximately 7 micrometers to approximately 4 micrometers. Stateddifferently, some of the peripheral area of the pixel surrounding theactive components is substantially reduced with respect to thedimensions given in connection with FIG. 10, without disturbing thetop-view and cross-sectional layout and design of the pixel's activecomponents as shown in FIGS. 10 and 11. A top view of such a pixel 105Ais shown in FIG. 20A, in which the dimension “e” is 5.1 micrometers andthe dimension “f” is 4.1 micrometers. In one aspect of this pixeldesign, to facilitate size reduction, fewer body connections B areincluded in the pixel 105A (e.g., one at each corner of the pixel) ascompared to the pixel shown in FIG. 10, which includes several bodyconnections B around the entire perimeter of the pixel.

As noted in FIG. 20, the array 100B includes 1348 columns 102 ₁ through102 ₁₃₄₈, wherein each column includes 1152 geometrically square pixels105A₁ through 105A₁₁₅₂, each having a size of approximately 5micrometers by 5 micrometers. Thus, the array includes over 1.5 millionpixels (>1.5 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 9millimeters by 9 millimeters. Like the array 100A of FIG. 19, in oneaspect the array 100B of FIG. 20 is divided into two groups of rows 400₁ and 400 ₂, as discussed above in connection with FIG. 19. In oneexemplary implementation, complete data frames (all pixels from both thefirst and second row groups 400 ₁ and 400 ₂) are acquired at a framerate of 50 frames/sec, thereby requiring 576 pairs of rows to besuccessively enabled for periods of approximately 35 microseconds each.For each enabled row, 674 pixels are read out by each column selectregister/output driver during approximately 31 microseconds (allowing 2microseconds at the beginning and end of each row, as discussed above inconnection with FIG. 18). Thus, in this example, each of the arrayoutput signals Vout1, Vout2, Vout3 and Vout4 has a data rate ofapproximately 22 MHz. Again, it should be appreciated that in otherimplementations, data may be acquired from the array 100B of FIG. 20 atframe rates other than 50 frames/sec.

FIG. 21 illustrates a block diagram of an ISFET sensor array 100C basedon a 0.35 micrometer CMOS fabrication process and incorporating thesmaller pixel size discussed above in connection with FIGS. 20 and 20A(5.1 micrometer square pixels), according to yet another embodiment. Asnoted in FIG. 21, the array 100C includes 4000 columns 102 ₁ through 102₄₀₀₀, wherein each column includes 3600 geometrically square pixels105A₁ through 105A₃₆₀₀, each having a size of approximately 5micrometers by 5 micrometers. Thus, the array includes over 14 millionpixels (>14 Mega-pixels) and, in one exemplary implementation, thecomplete array (ISFET pixels and associated circuitry) may be fabricatedas an integrated circuit chip having dimensions of approximately 22millimeters by 22 millimeters. Like the arrays 100A and 100B of FIGS. 19and 20, in one aspect the array 100C of FIG. 21 is divided into twogroups of rows 400 ₁ and 400 ₂. However, unlike the arrays 100A and100B, for each row group the array 100C includes sixteen column selectregisters and sixteen output drivers to simultaneously read sixteenpixels at a time in an enabled row, such that thirty-two output signalsVout1-Vout32 may be provided from the array 100C. In one exemplaryimplementation, complete data frames (all pixels from both the first andsecond row groups 400 ₁ and 400 ₂) may be acquired at a frame rate of 50frames/sec, thereby requiring 1800 pairs of rows to be successivelyenabled for periods of approximately 11 microseconds each. For eachenabled row, 250 pixels (4000/16) are read out by each column selectregister/output driver during approximately 7 microseconds (allowing 2microseconds at the beginning and end of each row). Thus, in thisexample, each of the array output signals Vout1-Vout32 has a data rateof approximately 35 MHz. As with the previous embodiments, it should beappreciated that in other implementations, data may be acquired from thearray 100C at frame rates other than 50 frames/sec.

While the exemplary arrays discussed above in connection with FIGS.13-21 are based on a 0.35 micrometer conventional CMOS fabricationprocess, it should be appreciated that arrays according to the presentdisclosure are not limited in this respect, as CMOS fabricationprocesses having feature sizes of less than 0.35 micrometers may beemployed (e.g., 0.18 micrometer CMOS processing techniques) to fabricatesuch arrays. Accordingly, ISFET sensor arrays with a pixel size/pitchsignificantly below 5 micrometers may be fabricated, providing forsignificantly denser ISFET arrays. For example, FIGS. 22 and 23illustrate respective block diagrams of ISFET sensor arrays 100D and100E according to yet other embodiments based on a 0.18 micrometer CMOSfabrication process, in which a pixel size of 2.6 micrometers isachieved. The pixel design itself is based substantially on the pixel105A shown in FIG. 20A, albeit on a smaller scale due to the 0.18micrometer CMOS process.

The array 100D of FIG. 22 includes 2800 columns 102 ₁ through 102 ₂₈₀₀,wherein each column includes 2400 geometrically square pixels eachhaving a size of approximately 2.6 micrometers by 2.6 micrometers. Thus,the array includes over 6.5 million pixels (>6.5 Mega-pixels) and, inone exemplary implementation, the complete array (ISFET pixels andassociated circuitry) may be fabricated as an integrated circuit chiphaving dimensions of approximately 9 millimeters by 9 millimeters. Likethe arrays 100A, 100B and 100C of FIGS. 19-21, in one aspect the array100D of FIG. 22 is divided into two groups of rows 400 ₁ and 400 ₂.However, unlike the arrays 100A, 100B, and 100C, for each row group thearray 100D includes eight column select registers and eight outputdrivers to simultaneously read eight pixels at a time in an enabled row,such that sixteen output signals Vout1-Vout16 may be provided from thearray 100D. In one exemplary implementation, complete data frames (allpixels from both the first and second row groups 400 ₁ and 400 ₂) may beacquired at a frame rate of 50 frames/sec, thereby requiring 1200 pairsof rows to be successively enabled for periods of approximately 16-17microseconds each. For each enabled row, 350 pixels (2800/8) are readout by each column select register/output driver during approximately 14microseconds (allowing 1 to 2 microseconds at the beginning and end ofeach row). Thus, in this example, each of the array output signalsVout1-Vout16 has a data rate of approximately 25 MHz. As with theprevious embodiments, it should be appreciated that in otherimplementations, data may be acquired from the array 100D at frame ratesother than 50 frames/sec.

The array 100E of FIG. 23 includes 7400 columns 102 ₁ through 102 ₇₄₀₀,wherein each column includes 7400 geometrically square pixels eachhaving a size of approximately 2.6 micrometers by 2.6 micrometers. Thus,the array includes over 54 million pixels (>54 Mega-pixels) and, in oneexemplary implementation, the complete array (ISFET pixels andassociated circuitry) may be fabricated as an integrated circuit chiphaving dimensions of approximately 21 millimeters by 21 millimeters.Like the arrays 100A-100D of FIGS. 19-22, in one aspect the array 100Eof FIG. 23 is divided into two groups of rows 400 ₁ and 400 ₂. However,unlike the arrays 100A-100D, for each row group the array 100E includesthirty-two column select registers and thirty-two output drivers tosimultaneously read thirty-two pixels at a time in an enabled row, suchthat sixty-four output signals Vout1-Vout64 may be provided from thearray 100E. In one exemplary implementation, complete data frames (allpixels from both the first and second row groups 400 ₁ and 400 ₂) may beacquired at a frame rate of 100 frames/sec, thereby requiring 3700 pairsof rows to be successively enabled for periods of approximately 3microseconds each. For each enabled row, 230 pixels (7400/32) are readout by each column select register/output driver during approximately700 nanoseconds. Thus, in this example, each of the array output signalsVout1-Vout64 has a data rate of approximately 328 MHz. As with theprevious embodiments, it should be appreciated that in otherimplementations, data may be acquired from the array 100D at frame ratesother than 100 frames/sec.

Thus, in various examples of ISFET arrays based on the inventiveconcepts disclosed herein, an array pitch of approximately nine (9)micrometers (e.g., a sensor surface area of less than ten micrometers byten micrometers) allows an ISFET array including over 256,000 pixels(i.e., a 512 by 512 array), together with associated row and columnselect and bias/readout electronics, to be fabricated on a 7 millimeterby 7 millimeter semiconductor die, and a similar sensor array includingover four million pixels (i.e., a 2048 by 2048 array, over 4Mega-pixels) to be fabricated on a 21 millimeter by 21 millimeter die.In other examples, an array pitch of approximately 5 micrometers allowsan ISFET array including approximately 1.55 Mega-pixels (i.e., a 1348 by1152 array) and associated electronics to be fabricated on a 9millimeter by 9 millimeter die, and an ISFET sensor array including over14 Mega-pixels and associated electronics on a 22 millimeter by 20millimeter die. In yet other implementations, using a CMOS fabricationprocess in which feature sizes of less than 0.35 micrometers arepossible (e.g., 0.18 micrometer CMOS processing techniques), ISFETsensor arrays with a pixel size/pitch significantly below 5 micrometersmay be fabricated (e.g., array pitch of 2.6 micrometers or pixel/sensorarea of less than 8 or 9 micrometers²), providing for significantlydense ISFET arrays.

In the embodiments of ISFET arrays discussed above, array pixels employa p-channel ISFET, as discussed above in connection with FIG. 9. Itshould be appreciated, however, that ISFET arrays according to thepresent disclosure are not limited in this respect, and that in otherembodiments pixel designs for ISFET arrays may be based on an n-channelISFET. In particular, any of the arrays discussed above in connectionwith FIGS. 13 and 19-23 may be implemented with pixels based onn-channel ISFETs.

For example, FIG. 24 illustrates the pixel design of FIG. 9 implementedwith an re-channel ISFET and accompanying n-channel MOSFETs, accordingto another inventive embodiment of the present disclosure. Morespecifically, FIG. 24 illustrates one exemplary pixel 105 ₁ of an arraycolumn (i.e., the first pixel of the column), together with columnbias/readout circuitry 110 j, in which the ISFET 150 (Q1) is ann-channel ISFET. Like the pixel design of FIG. 9, the pixel design ofFIG. 24 includes only three components, namely, the ISFET 150 and twon-channel MOSFET switches Q2 and Q3, responsive to one of n row selectsignals (RowSel₁ through RowSel_(n), logic high active). No transmissiongates are required in the pixel of FIG. 24, and all devices of the pixelare of a “same type,” i.e., n-channel devices. Also like the pixeldesign of FIG. 9, only four signal lines per pixel, namely the lines 112₁, 114 ₁, 116 ₁ and 118 ₁, are required to operate the three componentsof the pixel 105 ₁ shown in FIG. 24. In other respects, the pixeldesigns of FIG. 9 and FIG. 24 are similar, in that they are bothconfigured with a constant drain current I_(Dj) and a constantdrain-to-source voltage V_(DSj) to obtain an output signal V_(Sj) froman enabled pixel.

One of the primary differences between the n-channel ISFET pixel designof FIG. 24 and the p-channel ISFET design of FIG. 9 is the oppositedirection of current flow through the pixel. To this end, in FIG. 24,the element 106 is a controllable current sink coupled to the analogcircuitry supply voltage ground VSSA, and the element 108 _(j) of thebias/readout circuitry 110 j is a controllable current source coupled tothe analog positive supply voltage VDDA. Additionally, the bodyconnection of the ISFET 150 is not tied to its source, but rather to thebody connections of other ISFETs of the array, which in turn is coupledto the analog ground VSSA, as indicated in FIG. 24.

In addition to the pixel designs shown in FIGS. 9 and 24 (based on aconstant ISFET drain current and constant ISFET drain-source voltage),alternative pixel designs are contemplated for ISFET arrays, based onboth p-channel ISFETs and n-channel ISFETs, according to yet otherinventive embodiments of the present disclosure, as illustrated in FIGS.25-27. As discussed below, some alternative pixel designs may requireadditional and/or modified signals from the array controller 250 tofacilitate data acquisition. In particular, a common feature of thepixel designs shown in FIGS. 25-27 includes a sample and hold capacitorwithin each pixel itself, in addition to a sample and hold capacitor foreach column of the array. While the alternative pixel designs of FIGS.25-27 generally include a greater number of components than the pixeldesigns of FIGS. 9 and 24, the feature of a pixel sample and holdcapacitor enables “snapshot” types of arrays, in which all pixels of anarray may be enabled simultaneously to sample a complete frame andacquire signals representing measurements of one or more analytes inproximity to respective ISFETs of the array. In some applications, thismay provide for higher data acquisition speeds and/or improved signalsensitivity (e.g., higher signal-to-noise ratio).

FIG. 25 illustrates one such alternative design for a single pixel 105Cand associated column circuitry 110 j. The pixel 105C employs ann-channel ISFET and is based generally on the premise of providing aconstant voltage across the ISFET Q1 based on a feedback amplifier (Q4,Q5 and Q6). In particular, transistor Q4 constitutes the feedbackamplifier load, and the amplifier current is set by the bias voltage VB1(provided by the array controller). Transistor Q5 is a common gateamplifier and transistor Q6 is a common source amplifier. Again, thepurpose of feedback amplifier is to hold the voltage across the ISFET Q1constant by adjusting the current supplied by transistor Q3. TransistorQ2 limits the maximum current the ISFET Q1 can draw (e.g., so as toprevent damage from overheating a very large array of pixels). Thismaximum current is set by the bias voltage VB2 (also provided by thearray controller). In one aspect of the pixel design shown in FIG. 25,power to the pixel 105C may be turned off by setting the bias voltageVB2 to 0 Volts and the bias voltage VB1 to 3.3 Volts. In this manner,the power supplied to large arrays of such pixels may be modulated(turned on for a short time period and then off by the array controller)to obtain ion concentration measurements while at the same time reducingoverall power consumption of the array. Modulating power to the pixelsalso reduces heat dissipation of the array and potential heating of theanalyte solution, thereby also reducing any potentially deleteriouseffects from sample heating.

In FIG. 25, the output of the feedback amplifier (the gate of transistorQ3) is sampled by MOS switch Q7 and stored on a pixel sample and holdcapacitor Csh within the pixel itself. The switch Q7 is controlled by apixel sample and hold signal pSH (provided to the array chip by thearray controller), which is applied simultaneously to all pixels of thearray so as to simultaneously store the readings of all the pixels ontheir respective sample and hold capacitors. In this manner, arraysbased on the pixel design of FIG. 25 may be considered as “snapshot”arrays, in that a full frame of data is sampled at any given time,rather than sampling successive rows of the array. After each pixelvalue is stored on the corresponding pixel sample and hold capacitorCsh, each pixel 105C (ISFET and feedback amplifier) is free to acquireanother pH reading or it can by turned off to conserve power.

In FIG. 25, the pixel values stored on all of the pixel sample and holdcapacitors Csh are applied to the column circuitry 110 j one row at atime through source follower Q8, which is enabled via the transistor Q9in response to a row select signal (e.g., RowSel1). In particular, aftera row is selected and has settled out, the values stored in the pixelsample and hold capacitors are then in turn stored on the column sampleand hold capacitors Csh2, as enabled by the column sample and holdsignal COL SH, and provided as the column output signal V_(COLj).

FIG. 26 illustrates another alternative design for a single pixel 105Dand associated column circuitry 110 j, according to one embodiment ofthe present disclosure. In this embodiment, the ISFET is shown as ap-channel device. At the start of a data acquisition cycle, CMOSswitches controlled by the signals pSH (pixel sample/hold) and pRST(pixel reset) are closed (these signals are supplied by the arraycontroller). This pulls the source of ISFET (Q1) to the voltage VRST.Subsequently, the switch controlled by the signal pRST is opened, andthe source of ISFET Q1 pulls the pixel sample and hold capacitor Csh toa threshold below the level set by pH. The switch controlled by thesignal pSH is then opened, and the pixel output value is coupled, viaoperation of a switch responsive to the row select signal RowSel1, tothe column circuitry 110 j to provide the column output signal V_(COLj).Like the pixel design in the embodiment illustrated in FIG. 25, arraysbased on the pixel 105D are “snapshot” type arrays in that all pixels ofthe array may be operated simultaneously. In one aspect, this designallows a long simultaneous integration time on all pixels followed by ahigh-speed read out of an entire frame of data.

FIG. 27 illustrates yet another alternative design for a single pixel105E and associated column circuitry 110 j, according to one embodimentof the present disclosure. In this embodiment, again the ISFET is shownas a p-channel device. At the start of a data acquisition cycle, theswitches operated by the control signals p1 and pRST are briefly closed.This clears the value stored on the sampling capacitor Csh and allows acharge to be stored on ISFET (Q1). Subsequently, the switch controlledby the signal pSH is closed, allowing the charge stored on the ISFET Q1to be stored on the pixel sample and hold capacitor Csh. The switchcontrolled by the signal pSH is then opened, and the pixel output valueis coupled, via operation of a switch responsive to the row selectsignal RowSel1, to the column circuitry 110 j to provide the columnoutput signal V_(COLj). Gain may be provided in the pixel 105E via theratio of the ISFET capacitance to the Csh cap, i.e., gain=C_(Q1)/C_(sh),or by enabling the pixel multiple times (i.e., taking multiple samplesof the analyte measurement) and accumulating the ISFET output on thepixel sample and hold capacitor Csh without resetting the capacitor(i.e., gain is a function of the number of accumulations). Like theembodiments of FIGS. 25 and 26, arrays based on the pixel 105D are“snapshot” type arrays in that all pixels of the array may be operatedsimultaneously.

Turning from the sensor discussion, we will now be addressing thecombining of the ISFET array with a microwell array and the attendantfluidics. As most of the drawings of the microwell array structure arepresented only in cross-section or showing that array as only a block ina simplified diagram, FIGS. 28A and 28B are provided to assist thereader in beginning to visualize the resulting apparatus inthree-dimensions. FIG. 28A shows a group of round cylindrical wells 2810arranged in an array, while FIG. 28B shows a group of rectangularcylindrical wells 2830 arranged in an array. It will be seen that thewells are separated (isolated) from each other by the material 2840forming the well walls. While it is certainly possible to fabricatewells of other cross sections, in some embodiments it may not beadvantageous to do so. Such an array of microwells sits over theabove-discussed ISFET array, with one or more ISFETs per well. In thesubsequent drawings, when the microwell array is identified, one maypicture one of these arrays.

For many uses, to complete a system for sensing chemical reactions orchemical agents using the above-explained high density electronicarrays, techniques and apparatus are required for delivery to the arrayelements (called “pixels”) fluids containing chemical or biochemicalcomponents for sensing. In this section, exemplary techniques andmethods will be illustrated, which are useful for such purposes, withdesirable characteristics.

As high speed operation of the system may be desired, it is preferredthat the fluid delivery system, insofar as possible, not limit the speedof operation of the overall system.

Accordingly, needs exist not only for high-speed, high-density arrays ofISFETs or other elements sensitive to ion concentrations or otherchemical attributes, or changes in chemical attributes, but also forrelated mechanisms and techniques for supplying to the array elementsthe samples to be evaluated, in sufficiently small reaction volumes asto substantially advance the speed and quality of detection of thevariable to be sensed.

There are two and sometimes three components or subsystems, and relatedmethods, involved in delivery of the subject chemical samples to thearray elements: (1) macrofluidic system of reagent and wash fluidsupplies and appropriate valving and ancillary apparatus, (2) a flowcell and (3) in many applications, a microwell array. Each of thesesubsystems will be discussed, though in reverse order.

As discussed elsewhere, for many uses, such as in DNA sequencing, it isdesirable to provide over the array of semiconductor sensors acorresponding array of microwells, each microwell being small enoughpreferably to receive only one DNA-loaded bead, in connection with whichan underlying pixel in the array will provide a corresponding outputsignal.

The use of such a microwell array involves three stages of fabricationand preparation, each of which is discussed separately: (1) creating thearray of microwells to result in a chip having a coat comprising amicrowell array layer; (2) mounting of the coated chip to a fluidicinterface; and in the case of DNA sequencing, (3) loading DNA-loadedbead or beads into the wells. It will be understood, of course, that inother applications, beads may be unnecessary or beads having differentcharacteristics may be employed.

The systems described herein can include an array of microfluidicreaction chambers integrated with a semiconductor comprising an array ofchemFETs. In some embodiments, the invention encompasses such an array.The reaction chambers may, for example, be formed in a glass,dielectric, photodefineable or etchable material. The glass material maybe silicon dioxide.

Preferably, the array comprises at least 100,000 chambers. Preferably,each reaction chamber has a horizontal width and a vertical depth thathas an aspect ratio of about 1:1 or less. Preferably, the pitch betweenthe reaction chambers is no more than about 10 microns.

The above-described array can also be provided in a kit for sequencing.Thus, in some embodiments, the invention encompasses a kit comprising anarray of microfluidic reaction chambers integrated with an array ofchemFETs, and one or more amplification reagents.

In some embodiments, the invention encompasses a sequencing apparatuscomprising a dielectric layer overlying a chemFET, the dielectric layerhaving a recess laterally centered atop the chemFET. Preferably, thedielectric layer is formed of silicon dioxide.

Microwell fabrication may be accomplished in a number of ways. Theactual details of fabrication may require some experimentation and varywith the processing capabilities that are available.

In general, fabrication of a high density array of microwells involvesphoto-lithographically patterning the well array configuration on alayer or layers of material such as photoresist (organic or inorganic),a dielectric, using an etching process. The patterning may be done withthe material on the sensor array or it may be done separately and thentransferred onto the sensor array chip, of some combination of the two.However, techniques other than photolithography are not to be excludedif they provide acceptable results.

One example of a method for forming a microwell array is now discussed,starting with reference to FIG. 29. That figure diagrammatically depictsa top view of one corner (i.e., the lower left corner) of the layout ofa chip showing an array 2910 of the individual ISFET sensors 2912 on theCMOS die 2914. Signal lines 2916 and 2918 are used for addressing thearray and reading its output. Block 2920 represents some of theelectronics for the array, as discussed above, and layer 2922 representsa portion of a wall which becomes part of a microfluidics structure, theflow cell, as more fully explained below; the flow cell is thatstructure which provides a fluid flow over the microwell array or overthe sensor surface directly, if there is no microwell structure. On thesurface of the die, a pattern such as pattern 2922 at the bottom left ofFIG. 29 may be formed during the semiconductor processing to form theISFETs and associated circuitry, for use as alignment marks for locatingthe wells over the sensor pixels when the dielectric has covered thedie's surface.

After the semiconductor structures, as shown, are formed, the microwellstructure is applied to the die. That is, the microwell structure can beformed right on the die or it may be formed separately and then mountedonto the die, either approach being acceptable. To form the microwellstructure on the die, various processes may be used. For example, theentire die may be spin-coated with, for example, a negative photoresistsuch as Microchem's SU-8 2015 or a positive resist/polyimide such as HDMicrosystems HD8820, to the desired height of the microwells. Thedesired height of the wells (e.g., about 4-12 μm in the example of onepixel per well, though not so limited as a general matter) in thephotoresist layer(s) can be achieved by spinning the appropriate resistat predetermined rates (which can be found by reference to theliterature and manufacturer specifications, or empirically), in one ormore layers. (Well height typically may be selected in correspondencewith the lateral dimension of the sensor pixel, preferably for a nominal1:1-1.5:1 aspect ratio, height:width or diameter. Based onsignal-to-noise considerations, there is a relationship betweendimensions and the required data sampling rates to achieve a desiredlevel of performance. Thus there are a number of factors that will gointo selecting optimum parameters for a given application.)Alternatively, multiple layers of different photoresists may be appliedor another form of dielectric material may be deposited. Various typesof chemical vapor deposition may also be used to build up a layer ofmaterials suitable for microwell formation therein.

Once the photoresist layer (the singular form “layer” is used toencompass multiple layers in the aggregate, as well) is in place, theindividual wells (typically mapped to have either one or four ISFETsensors per well) may be generated by placing a mask (e.g., of chromium)over the resist-coated die and exposing the resist to cross-linking(typically UV) radiation. All resist exposed to the radiation (i.e.,where the mask does not block the radiation) becomes cross-linked and asa result will form a permanent plastic layer bonded to the surface ofthe chip (die). Unreacted resist (i.e., resist in areas which are notexposed, due to the mask blocking the light from reaching the resist andpreventing cross-linking) is removed by washing the chip in a suitablesolvent (i.e., developer) such as propyleneglycolmethylethylacetate(PGMEA) or other appropriate solvent. The resultant structure definesthe walls of the microwell array.

FIG. 30 shows an example of a layout for a portion of a chromium mask3010 for a one-sensor-per-well embodiment, corresponding to the portionof the die shown in FIG. 29. The grayed areas 3012, 3014 are those thatblock the UV radiation. The alignment marks in the white portions 3016on the bottom left quadrant of FIG. 30, within gray area 3012, are usedto align the layout of the wells with the ISFET sensors on the chipsurface. The array of circles 3014 in the upper right quadrant of themask block radiation from reaching the well areas, to leave unreactedresist which can be dissolved in forming the wells.

FIG. 31 shows a corresponding layout for the mask 3020 for a4-sensors-per-well embodiment. Note that the alignment pattern 3016 isstill used and that the individual well-masking circles 3014A in thearray 2910 now have twice the diameter as the wells 3014 in FIG. 30, foraccommodating four sensors per well instead of one sensor-per-well.

After exposure of the die/resist to the UV radiation, a second layer ofresist may be coated on the surface of the chip. This layer of resistmay be relatively thick, such as about 400-450 μm thick, typically. Asecond mask 3210 (FIG. 32), which also may be of chromium, is used tomask an area 3220 which surrounds the array, to build a collar or wall(or basin, using that term in the geological sense) 3310 of resist whichsurrounds the active array of sensors on substrate 3312, as shown inFIG. 33. In the particular example being described, the collar is 150 μmwider than the sensor array, on each side of the array, in the xdirection, and 9 μm wider on each side than the sensor array, in the ydirection. Alignment marks on mask 3210 (most not shown) are matched upwith the alignment marks on the first layer and the CMOS chip itself.

Other photolithographic approaches may be used for formation of themicrowell array, of course, the foregoing being only one example.

For example, contact lithography of various resolutions and with variousetchants and developers may be employed. Both organic and inorganicmaterials may be used for the layer(s) in which the microwells areformed. The layer(s) may be etched on a chip having a dielectric layerover the pixel structures in the sensor array, such as a passivationlayer, or the layer(s) may be formed separately and then applied overthe sensor array. The specific choice or processes will depend onfactors such as array size, well size, the fabrication facility that isavailable, acceptable costs, and the like.

Among the various organic materials which may be used in someembodiments to form the microwell layer(s) are the above-mentioned SU-8type of negative-acting photoresist, a conventional positive-actingphotoresist and a positive-acting photodefineable polyimide. Each hasits virtues and its drawbacks, well known to those familiar with thephotolithographic art.

Naturally, in a production environment, modifications will beappropriate.

Contact lithography has its limitations and it may not be the productionmethod of choice to produce the highest densities of wells—i.e., it mayimpose a higher than desired minimum pitch limit in the lateraldirections. Other techniques, such as a deep UV step-and-repeat process,are capable of providing higher resolution lithography and can be usedto produce small pitches and possibly smaller well diameters. Of course,for different desired specifications (e.g., numbers of sensors and wellsper chip), different techniques may prove optimal. And pragmaticfactors, such as the fabrication processes available to a manufacturer,may motivate the use of a specific fabrication method. While novelmethods are discussed, various aspects of the invention are limited touse of these novel methods.

Preferably the CMOS wafer with the ISFET array will be planarized afterthe final metallization process. A chemical mechanical dielectricplanarization prior to the silicon nitride passivation is suitable. Thiswill allow subsequent lithographic steps to be done on very flatsurfaces which are free of back-end CMOS topography.

By utilizing deep-UV step-and-repeat lithography systems, it is possibleto resolve small features with superior resolution, registration, andrepeatability. However, the high resolution and large numerical aperture(NA) of these systems precludes their having a large depth of focus. Assuch, it may be necessary, when using such a fabrication system, to usethinner photodefinable spin-on layers (i.e., resists on the order of 1-2μm rather than the thicker layers used in contact lithography) topattern transfer and then etch microwell features to underlying layer orlayers. High resolution lithography can then be used to pattern themicrowell features and conventional SiO₂ etch chemistries can beused—one each for the bondpad areas and then the microwell areas—havingselective etch stops; the etch stops then can be on aluminum bondpadsand silicon nitride passivation (or the like), respectively.Alternatively, other suitable substitute pattern transfer and etchprocesses can be employed to render microwells of inorganic materials.

Another approach is to form the microwell structure in an organicmaterial. For example, a dual-resist “soft-mask” process may beemployed, whereby a thin high-resolution deep-UV resist is used on topof a thicker organic material (e.g., cured polyimide or opposite-actingresist). The top resist layer is patterned. The pattern can betransferred using an oxygen plasma reactive ion etch process. Thisprocess sequence is sometimes referred to as the “portable conformablemask” (PCM) technique. See B. J. Lin et al., “Practicing the Novolacdeep-UV portable conformable masking technique”, Journal of VacuumScience and Technology 19, No. 4, 1313-1319 (1981); and A. Cooper et al,“Optimization of a photosensitive spin-on dielectric process for copperinductor coil and interconnect protection in RF SoC devices.”

Alternatively a “drill-focusing” technique may be employed, wherebyseveral sequential step-and-repeat exposures are done at different focaldepths to compensate for the limited depth of focus (DOF) ofhigh-resolution steppers when patterning thick resist layers. Thistechnique depends on the stepper NA and DOF as well as the contrastproperties of the resist material.

Another PCM technique may be adapted to these purposes, such as thatshown in U.S. patent application publication no. 2006/0073422 by Edwardset al. This is a three-layer PCM process and it is illustrated in FIG.33A. As shown there, basically six major steps are required to producethe microwell array and the result is quite similar to what contactlithography would yield.

In a first step, 3320, a layer of high contrast negative-actingphotoresist such as type Shipley InterVia Photodielectric Material 8021(IV8021) 3322 is spun on the surface of a wafer, which we shall assumeto be the wafer providing the substrate 3312 of FIG. 33 (in which thesensor array is fabricated), and a soft bake operation is performed.Next, in step 3324, a blocking anti-reflective coating (BARC) layer3326, is applied and soft baked. On top of this structure, a thin resistlayer 3328 is spun on and soft baked, step 3330, the thin layer ofresist being suitable for fine feature definition. The resist layer 3328is then patterned, exposed and developed, and the BARC in the exposedregions 3329, not protected any longer by the resist 3328, is removed,Step 3332. This opens up regions 3329 down to the uncured IV8021 layer.The BARC layer can now act like a conformal contact mask A blanketexposure with a flooding exposure tool, Step 3334, cross-links theexposed IV8021, which is now shown as distinct from the uncured IV8021at 3322. One or more developer steps 3338 are then performed, removingeverything but the cross-linked IV8021 in regions 3336. Regions 3336 nowconstitute the walls of the microwells.

Although as shown above, the wells bottom out (i.e. terminate) on thetop passivation layer of the ISFETs, it is believed that an improvementin ISFET sensor performance (i.e. such as signal-to-noise ratio) can beobtained if the active bead(s) is(are) kept slightly elevated from theISFET passivation layer. One way to do so is to place a spacer “bump”within the boundary of the pixel microwell. An example of how this couldbe rendered would be not etching away a portion of the layer-or-layersused to form the microwell structure (i.e. two lithographic steps toform the microwells—one to etch part way done, the other to pattern thebump and finish the etch to bottom out), by depositing andlithographically defining and etching a separate layer to form the“bump”, by using a permanent photo-definable material for the bump oncethe microwells are complete, or by forming the bump prior to forming themicrowell. The bump feature is shown as 3350 in FIG. 33B. An alternative(or additional) non-integrated approach is to load the wells with alayer or two of very small packing beads before loading the DNA-bearingbeads.

Using a 6 um (micron) thick layer of tetra-methyl-ortho-silicate (TEOS)as a SiO₂-like layer for microwell formation, FIG. 33B-1 shows ascanning electron microscope (SEM) image of a cross-section of a portion3300A of an array architecture as taught herein. Microwells 3302A areformed in the TEOS layer 3304A. The wells extend about 4 um into the 6um thick layer. Typically, the etched well bottoms on an etch-stopmaterial which may be, for example, an oxide, an organic material orother suitable material known in semiconductor processing foretch-stopping use. A thin layer of etch stop material may be formed ontop of a thicker layer of polyimide or other suitable dielectric, suchthat there is about 2 um of etch stop+polyimide between the well bottomand the Metal4 (M4) layer of the chip in which the extended gateelectrode 3308A is formed for each underlying ISFET in the array. Aslabeled on the side, the CMOS metallization layers M3, M2 and M1, whichform lower level interconnects and structures, are shown, with the ISFETchannels being formed in the areas indicated by arrows 3310A.

In the orthogonal cross-sectional view (i.e., looking down from thetop), the wells may be formed in either round or square shape. Roundwells may improve bead capture and may obviate the need for packingbeads at the bottom or top of the wells.

The tapered slopes to the sides of the microwells also may be used toadvantage. Referring to FIG. 33B-2, if the beads 3320A have a diameterlarger than the bottom span across the wells, but small enough to fitinto the mouths of the wells, the beads will be spaced off the bottom ofthe wells due to the geometric constraints. For example, FIG. 33B-2illustrates the example of microwells that are square in cross-sectionas viewed from the top, 4 um on a side, with 3.8 um diameter beads 3320Aloaded. Experimentally and with some calculation, one may determinesuitable bead size and well dimension combinations. FIG. 33B-3 shows aportion of one 4 um well loaded with a 2.8 um diameter bead 3322A, whichobviously is relatively small and falls all the way to the bottom of thewell; a 4.0 um diameter bead 3324A which is stopped from reaching thebottom by the sidewall taper of the well; and an intermediate-sized bead3326A of 3.6 um diameter which is spaced from the well bottom by packingbeads 3328A. Clearly, bead size has to be carefully matched to themicrowell etch taper.

Thus, microwells can be fabricated by any high aspect ratiophoto-definable or etchable thin-film process, that can providerequisite thickness (e.g., about 4-10 um). Among the materials believedto be suitable are photosensitive polymers, deposited silicon dioxide,non-photosensitive polymer which can be etched using, for example,plasma etching processes, etc. In the silicon dioxide family, TEOS andsilane nitrous oxide (SILOX) appear suitable. The final structures aresimilar but the various materials present differing surface compositionsthat may cause the target biology or chemistry to react differently.

When the microwell layer is formed, it may be necessary to provide anetch stop layer so that the etching process does not proceed furtherthan desired. For example, there may be an underlying layer to bepreserved, such as a low-K dielectric. The etch stop material should beselected according to the application. SiC and SiN materials may besuitable, but that is not meant to indicate that other materials may notbe employed, instead. These etch-stop materials can also serve toenhance the surface chemistry which drives the ISFET sensor sensitivity,by choosing the etch-stop material to have an appropriate point of zerocharge (PZC). Various metal oxides may be suitable addition to silicondioxide and silicon nitride.

The PZCs for various metal oxides may be found in various texts, such as“Metal Oxides—Chemistry and Applications” by J. Fierro. We have learnedthat Ta₂O₅ may be preferred as an etch stop over Al₂O₃ because the PZCof Al₂O₃ is right at the pH being used (i.e., about 8.8) and, hence,right at the point of zero charge. In addition Ta₂O₅ has a highersensitivity to pH (i.e., mV/pH), another important factor in the sensorperformance. Optimizing these parameters may require judicious selectionof passivation surface materials.

Using thin metal oxide materials for this purpose (i.e., as an etch stoplayer) is difficult due to the fact of their being so thinly deposited(typically 200-500 A). A post-microwell fabrication metal oxidedeposition technique may allow placement of appropriate PZC metal oxidefilms at the bottom of the high aspect ratio microwells.

Electron-beam depositions of (a) reactively sputtered tantalum oxide,(b) non-reactive stoichiometric tantalum oxide, (c) tungsten oxide, or(d) Vanadium oxide may prove to have superior “down-in-well” coveragedue to the superior directionality of the deposition process.

The array typically comprises at least 100 microfluidic wells, each ofwhich is coupled to one or more chemFET sensors. Preferably, the wellsare formed in at least one of a glass (e.g., SiO₂), a polymericmaterial, a photodefinable material or a reactively ion etchable thinfilm material. Preferably, the wells have a width to height ratio lessthan about 1:1. Preferably the sensor is a field effect transistor, andmore preferably a chemFET. The chemFET may optionally be coupled to aPPi receptor. Preferably, each of the chemFETs occupies an area of thearray that is 10² microns or less.

In some embodiments, the invention encompasses a sequencing devicecomprising a semiconductor wafer device coupled to a dielectric layersuch as a glass (e.g., SiO₂), polymeric, photodefinable or reactive ionetchable material in which reaction chambers are formed. Typically, theglass, dielectric, polymeric, photodefinable or reactive ion etchablematerial is integrated with the semiconductor wafer layer. In someinstances, the glass, polymeric, photodefinable or reactive ion etchablelayer is non-crystalline. In some instances, the glass may be SiO₂. Thedevice can optionally further comprise a fluid delivery module of asuitable material such as a polymeric material, preferably an injectionmoldable material. More preferably, the polymeric layer ispolycarbonate.

In some embodiments, the invention encompasses a method formanufacturing a sequencing device comprising: using photolithography,generating wells in a glass, dielectric, photodefinable or reactivelyion etchable material on top of an array of transistors.

The process of using the assembly of an array of sensors on a chipcombined with an array of microwells to sequence the DNA in a sample isreferred to as an “experiment.” Executing an experiment requires loadingthe wells with the DNA-bound beads and the flowing of several differentfluid solutions (i.e., reagents and washes) across the wells. A fluiddelivery system (e.g., valves, conduits, pressure source(s), etc.)coupled with a fluidic interface is needed which flows the varioussolutions across the wells in a controlled even flow with acceptablysmall dead volumes and small cross contamination between sequentialsolutions. Ideally, the fluidic interface to the chip (sometimesreferred to as a “flow cell”) would cause the fluid to reach allmicrowells at the same time. To maximize array speed, it is necessarythat the array outputs be available at as close to the same time aspossible. The ideal clearly is not possible, but it is desirable tominimize the differentials, or skews, of the arrival times of anintroduced fluid, at the various wells, in order to maximize the overallspeed of acquisition of all the signals from the array.

Flow cell designs of many configurations are possible; thus the systemand methods presented herein are not dependent on use of a specific flowcell configuration. It is desirable, though, that a suitable flow cellsubstantially conform to the following set of objectives:

have connections suitable for interconnecting with a fluidics deliverysystem—e.g., via appropriately-sized tubing;

have appropriate head space above wells;

minimize dead volumes encountered by fluids;

minimize small spaces in contact with liquid but not quickly swept cleanby flow of a wash fluid through the flow cell (to minimize crosscontamination);

be configured to achieve uniform transit time of the flow over thearray;

generate or propagate minimal bubbles in the flow over the wells;

be adaptable to placement of a removable reference electrode inside oras close to the flow chamber as possible;

facilitate easy loading of beads;

be manufacturable at acceptable cost; and

be easily assembled and attached to the chip package.

Satisfaction of these criteria so far as possible will contribute tosystem performance positively. For example, minimization of bubbles isimportant so that signals from the array truly indicate the reaction ina well rather than being spurious noise.

Each of several example designs will be discussed, meeting thesecriteria in differing ways and degrees. In each instance, one typicallymay choose to implement the design in one of two ways: either byattaching the flow cell to a frame and gluing the frame (or otherwiseattaching it) to the chip or by integrating the frame into the flow cellstructure and attaching this unified assembly to the chip. Further,designs may be categorized by the way the reference electrode isintegrated into the arrangement. Depending on the design, the referenceelectrode may be integrated into the flow cell (e.g., form part of theceiling of the flow chamber) or be in the flow path (typically to theoutlet or downstream side of the flow path, after the sensor array).

A first example of a suitable experiment apparatus 3410 incorporatingsuch a fluidic interface is shown in FIGS. 34-37, the manufacture andconstruction of which will be discussed in greater detail below.

The apparatus comprises a semiconductor chip 3412 (indicated generally,though hidden) on or in which the arrays of wells and sensors areformed, and a fluidics assembly 3414 on top of the chip and deliveringthe sample to the chip for reading. The fluidics assembly includes aportion 3416 for introducing fluid containing the sample, a portion 3418for allowing the fluid to be piped out, and a flow chamber portion 3420for allowing the fluid to flow from inlet to outlet and along the wayinteract with the material in the wells. Those three portions areunified by an interface comprising a glass slide 3422 (e.g., ErieMicroarray Cat #C22-5128-M20 from Erie Scientific Company, Portsmouth,N.H., cut in thirds, each to be of size about 25 mm×25 mm).

Mounted on the top face of the glass slide are two fittings, 3424 and3426, such as nanoport fittings Part #N-333 from Upchurch Scientific ofOak Harbor, Wash. One port (e.g., 3424) serves as an inlet deliveringliquids from the pumping/valving system described below but not shownhere. The second port (e.g., 3426) is the outlet which pipes the liquidsto waste. Each port connects to a conduit 3428, 3432 such as flexibletubing of appropriate inner diameter. The nanoports are mounted suchthat the tubing can penetrate corresponding holes in the glass slide.The tube apertures should be flush with the bottom surface of the slide.

On the bottom of the glass slide, flow chamber 3420 may comprise variousstructures for promoting a substantially laminar flow across themicrowell array. For example, a series of microfluidic channels fanningout from the inlet pipe to the edge of the flow chamber may be patternedby contact lithography using positive photoresists such as SU-8photoresist from MicroChem. Corp. of Newton, Mass. Other structures willbe discussed below.

The chip 3412 will in turn be mounted to a carrier 3430, for packagingand connection to connector pins 3432.

For ease of description, to discuss fabrication starting with FIG. 38 weshall now consider the glass slide 3422 to be turned upside downrelative to the orientation it has in FIGS. 34-37.

A layer of photoresist 3810 is applied to the “top” of the slide (whichwill become the “bottom” side when the slide and its additional layersis turned over and mounted to the sensor assembly of ISFET array withmicrowell array on it). Layer 3810 may be about 150 μm thick in thisexample, and it will form the primary fluid carrying layer from the endof the tubing in the nanoports to the edge of the sensor array chip.Layer 3810 is patterned using a mask such as the mask 3910 of FIG. 39(“patterned” meaning that a radiation source is used to expose theresist through the mask and then the non-plasticized resist is removed).The mask 3910 has radiation-transparent regions which are shown as whiteand radiation-blocking regions 3920, which are shown in shading. Theradiation-blocking regions are at 3922-3928. The region 3926 will form achannel around the sensor assembly; it is formed about 0.5 mm inside theouter boundary of the mask 3920, to avoid the edge bead that is typical.The regions 3922 and 3924 will block radiation so that correspondingportions of the resist are removed to form voids shaped as shown. Eachof regions 3922, 3924 has a rounded end dimensioned to receive an end ofa corresponding one of the tubes 3428, 3432 passing through acorresponding nanoport 3424, 3426. From the rounded end, the regions3922, 3924 fan out in the direction of the sensor array to allow theliquid to spread so that the flow across the array will be substantiallylaminar. The region 3928 is simply an alignment pattern and may be anysuitable alignment pattern or be replaced by a suitable substitutealignment mechanism. Dashed lines on FIG. 38 have been provided toillustrate the formation of the voids 3822 and 3824 under mask regions3922 and 3924.

A second layer of photoresist is formed quite separately, not on theresist 3810 or slide 3422. Preferably it is formed on a flat, flexiblesurface (not shown), to create a peel-off, patterned plastic layer. Asshown in FIG. 40, this second layer of photoresist may be formed using amask such as mask 4010, which will leave on the flexible substrate,after patterning, the border under region 4012, two slits under regions4014, 4016, whose use will be discussed below, and alignment marksproduced by patterned regions 4018 and 4022. The second layer ofphotoresist is then applied to the first layer of photoresist using onealignment mark or set of alignment marks, let's say produced by pattern4018, for alignment of these layers. Then the second layer is peeledfrom its flexible substrate and the latter is removed.

The other alignment mark or set of marks produced by pattern 4022 isused for alignment with a subsequent layer to be discussed.

The second layer is preferably about 150 μm deep and it will cover thefluid-carrying channel with the exception of a slit about 150 μm long ateach respective edge of the sensor array chip, under slit-formingregions 4014 and 4016.

Once the second layer of photoresist is disposed on the first layer, athird patterned layer of photoresist is formed over the second layer,using a mask such as mask 4110, shown in FIG. 41. The third layerprovides a baffle member under region 4112 which is as wide as thecollar 3310 on the sensor chip array (see FIG. 33) but about 300 μmnarrower to allow overlap with the fluid-carrying channel of the firstlayer. The third layer may be about 150 μm thick and will penetrate thechip collar 3310, toward the floor of the basin formed thereby, by 150μm. This configuration will leave a headspace of about 300 μm above thewells on the sensor array chip. The liquids are flowed across the wellsalong the entire width of the sensor array through the 150 μm slitsunder 4014, 4016.

FIG. 36 shows a partial sectional view, in perspective, of theabove-described example embodiment of a microfluidics and sensorassembly, also depicted in FIGS. 34 and 35, enlarged to make morevisible the fluid flow path. (A further enlarged schematic of half ofthe flow path is shown in FIG. 37.) Here, it will be seen that fluidenters via the inlet pipe 3428 in inlet port 3424. At the bottom of pipe3428, the fluid flows through the flow expansion chamber 3610 formed bymask area 3922, that the fluid flows over the collar 3310 and then downinto the bottom 3320 of the basin, and across the die 3412 with itsmicrowell array. After passing over the array, the fluid then takes avertical turn at the far wall of the collar 3310 and flows over the topof the collar to and across the flow concentration chamber 3612 formedby mask area 3924, exiting via outlet pipe 3432 in outlet port 3426.Part of this flow, from the middle of the array to the outlet, may beseen also in the enlarged diagrammatic illustration of FIG. 37, whereinthe arrows indicate the flow of the fluid.

The fluidics assembly may be secured to the sensor array chip assemblyby applying an adhesive to parts of mating surfaces of those twoassemblies, and pressing them together, in alignment.

Though not illustrated in FIGS. 34-36, the reference electrode may beunderstood to be a metallization 3710, as shown in FIG. 37, at theceiling of the flow chamber.

Another way to introduce the reference electrode is shown in FIG. 42.There, a hole 4210 is provided in the ceiling of the flow chamber and agrommet 4212 (e.g., of silicone) is fitted into that hole, providing acentral passage or bore through which a reference electrode 4220 may beinserted. Baffles or other microfeatures (not shown in FIG. 42 butdiscussed below in connection with FIG. 42A) may be patterned into theflow channel to promote laminar flow over the microwell array.

Achieving a uniform flow front and eliminating problematic flow pathareas is desirable for a number of reasons. One reason is that very fasttransition of fluid interfaces within the system's flow cell is desiredfor many applications, particularly gene sequencing. In other words, anincoming fluid must completely displace the previous fluid in a shortperiod of time. Uneven fluid velocities and diffusion within the flowcell, as well as problematic flow paths, can compete with thisrequirement. Simple flow through a conduit of rectangular cross sectioncan exhibit considerable disparity of fluid velocity from regions nearthe center of the flow volume to those adjacent the sidewalls, onesidewall being the top surface of the microwell layer and the fluid inthe wells. Such disparity leads to spatially and temporally largeconcentration gradients between the two traveling fluids. Further,bubbles are likely to be trapped or created in stagnant areas like sharpcorners interior the flow cell. (The surface energy (hydrophilic vs.hydrophobic) can significantly affect bubble retention. Avoidance ofsurface contamination during processing and use of a surface treatmentto create a more hydrophilic surface should be considered if theas-molded surface is too hydrophobic.) Of course, the physicalarrangement of the flow chamber is probably the factor which mostinfluences the degree of uniformity achievable for the flow front.

One approach is to configure the flow cross section of the flow chamberto achieve flow rates that vary across the array width so that thetransit times are uniform across the array. For example, the crosssection of the diffuser (i.e., flow expansion chamber) section 3416,3610 may be made as shown at 4204A in FIG. 42A, instead of simply beingrectangular, as at 4204A. That is, it may have a curved (e.g., concave)wall. The non-flat wall 4206A of the diffuser can be the top or thebottom. Another approach is to configure the effective path lengths intothe array so that the total path lengths from entrance to exit over thearray are essentially the same. This may be achieved, for example, byplacing flow-disrupting features such as cylinders or other structuresoriented normal to the flow direction, in the path of the flow. If theflow chamber has as a floor the top of the microwell array and as aceiling an opposing wall, these flow-disrupting structures may beprovided either on the top of the microwell layer or on (or in) theceiling wall. The structures must project sufficiently into the flow tohave the desired effect, but even small flow disturbances can havesignificant impact. Turning to FIGS. 42B-42F, there are showndiagrammatically some examples of such structures. In FIG. 42B, on thesurface of microwell layer 4210B there are formed a series ofcylindrical flow disruptors 4214B extending vertically toward the flowchamber ceiling wall 4212B, and serving to disturb laminar flow for thefluid moving in the direction of arrow A. FIG. 42C depicts a similararrangement except that the flow disruptors 4216C have rounded tops andappear more like bumps, perhaps hemispheres or cylinders with sphericaltops. By contrast, in FIG. 42D, the flow disruptors 4218D protrude, ordepend, from the ceiling wall 4212B of the flow chamber. Only one columnof flow disruptors is shown but it will be appreciated that a pluralityof more or less parallel columns typically would be required. Forexample, FIG. 42E shows several columns 4202E of such flow disruptors(projecting outwardly from ceiling wall 4212B (though the orientation isupside down relative to FIGS. 42B-42D). The spacing between thedisruptors and their heights may be selected to influence the distanceover which the flow profile becomes parabolic, so that transit timeequilibrates.

Another configuration, shown in FIGS. 42F and 42F1, involves the use ofsolid, beam-like projections or baffles 4220F as disruptors. Thisconcept may be used to form a ceiling member for the flow chamber. Suchan arrangement encourages more even fluid flow and significantly reducesfluid displacement times as compared with a simple rectangularcross-section without disruptor structure. Further, instead of fluidentry to the array occurring along one edge, fluid may be introduced atone corner 4242F, through a small port, and may exit from the oppositecorner, 4244F, via a port in fluid communication with that corner area.The series of parallel baffles 4220F separates the flow volume betweeninput and outlet corners into a series of channels. The lowest fluidresistant path is along the edge of the chip, perpendicular to thebaffles. As incoming liquid fills this channel, the fluid is thendirected between the baffles to the opposite side of the chip. Thechannel depth between each baffle pair preferably is graded across thechip, such that the flow is encouraged to travel toward the exit portthrough the farthest channel, thereby evening the flow front between thebaffles. The baffles extend downwardly nearly to the chip (i.e.,microwell layer) surface, but because they are quite thin, fluid candiffuse under them quickly and expose the associated area of the arrayassembly.

FIGS. 42F2-42F8 illustrate an example of a single-piece,injection-molded (preferably of polycarbonate) flow cell member 42F200which may be used to provide baffles 4220F, a ceiling to the flowchamber, fluid inlet and outlet ports and even the reference electrode.FIG. 42F7 shows an enlarged view of the baffles on the bottom of member42F200 and the baffles are shown as part of the underside of member42F200 in FIG. 42F6. As it is difficult to form rectangular features insmall dimensions by injection molding, the particular instance of thesebaffles, shown as 4220F′, are triangular in cross section.

In FIG. 42F2, there is a top, isometric view of member 42F200 mountedonto a sensor array package 42F300, with a seal 42F202 formed betweenthem and contact pins 42F204 depending from the sensor array chippackage. FIGS. 42F3 and 42F4 show sections, respectively, throughsection lines H-H and I-I of FIG. 42F5, permitting one to see inrelationship the sensor array chip 42F250, the baffles 4220F′ and fluidflow paths via inlet 42F260 and outlet 42F270 ports.

By applying a metallization to bottom 42F280 of member 42F200, thereference electrode may be formed.

Various other locations and approaches may be used for introducing fluidflow into the flow chamber, as well. In addition to embodiments in whichfluid may be introduced across the width of an edge of the chip assembly42F1, as in FIGS. 57-58, for example, or fluid may be introduced at onecorner of the chip assembly, as in FIG. 42F1. Fluid also may beintroduced, for example, as in FIGS. 42G and 42H, where fluid is flowedthrough an inlet conduit 4252G to be discharged adjacent and toward thecenter of the chip, as at 4254G, and flowed radially outwardly from theintroduction point.

FIGS. 42I and 42J in conjunction with FIGS. 42G and 42H depict incross-section an example of such a structure and its operation. Incontrast with earlier examples, this embodiment contains an additionalelement, a diaphragm valve, 4260I. Initially, as shown in FIG. 42H, thevalve 4260I is open, providing a path via conduit 4262I to a wastereservoir (not shown). The open valve provides a low impedance flow tothe waste reservoir or outlet. Air pressure is then applied to thediaphragm valve, as in FIG. 42J, closing the low impedance path andcausing the fluid flow to continue downwardly through central bore 4264Jin member 4266J which forms the ceiling of the flow chamber, and acrossthe chip (sensor) assembly. The flow is collected by the channels at theedges of the sensor, as described above, and exits to the waste outputvia conduit 4268J.

A variation on this idea is depicted in FIGS. 42K-42M, which show fluidbeing introduced not at the center of the chip assembly, but at onecorner, 4272K, instead. It flows across the chip 3412 as symbolicallyindicated by lines 4274K and is removed at the diagonally opposingcorner, 4276K. The advantage of this concept is that it all buteliminates any stagnation points. It also has the advantage that thesensor array can be positioned vertically so that the flow is introducedat the bottom and removed at the top to aid in the clearance of bubbles.This type of embodiment, by the way, may be considered as one quadrantof the embodiments with the flow introduced in the center of the array.An example of an implementation with a valve 4278L closed and shuntingflow to the waste outlet or reservoir is shown in FIG. 42L. The maindifference with respect to the embodiment of FIGS. 42I and 42J is thatthe fluid flow is introduced at a corner of the array rather than at itscenter.

In all cases, attention should be given to assuring a thorough washingof the entire flow chamber, along with the microwells, between reagentcycles. Flow disturbances may exacerbate the challenge of fully cleaningout the flow chamber.

Flow disturbances may also induce or multiply bubbles in the fluid. Abubble may prevent the fluid from reaching a microwell, or delay itsintroduction to the microwell, introducing error into the microwellreading or making the output from that microwell useless in theprocessing of outputs from the array. Thus, care should be taken inselecting configurations and dimensions for the flow disruptor elementsto manage these potential adverse factors. For example, a tradeoff maybe made between the heights of the disruptor elements and the velocityprofile change that is desired.

FIGS. 43-44 show another alternative flow cell design, 4310. This designrelies on the molding of a single plastic piece or member 4320 to beattached to the chip to complete the flow cell. The connection to thefluidic system is made via threaded connections tapped into appropriateholes in the plastic piece at 4330 and 4340. Or, if the member 4320 ismade of a material such as polydimethylsiloxane (PDMS), the connectionsmay be made by simply inserting the tubing into an appropriately sizedhole in the member 4320. A vertical cross section of this design isshown in FIGS. 43-44. This design may use an overhanging plastic collar4350 (which may be a solid wall as shown or a series of depending,spaced apart legs forming a downwardly extending fence-like wall) toenclose the chip package and align the plastic piece with the chippackage, or other suitable structure, and thereby to alignment the chipframe with the flow cell forming member 4320. Liquid is directed intothe flow cell via one of apertures 4330, 4340, thence downwardly towardsthe flow chamber.

In the illustrated embodiment, the reference electrode is introduced tothe top of the flow chamber via a bore 4325 in the member 4320. Theplacement of the removable reference electrode is facilitated by asilicone sleeve 4360 and an epoxy stop ring 4370 (see the blow-up ofFIG. 44). The silicone sleeve provides a tight seal and the epoxy stopring prevent the electrode from being inserted too far into the flowcell. Of course, other mechanisms may be employed for the same purposes,and it may not be necessary to employ structure to stop the electrode.And if a material such as PDMS is used for member 4320, the materialitself may form a watertight seal when the electrode is inserted,obviating need for the silicone sleeve.

FIGS. 45 and 46 show a similar arrangement except that member 4510 lacksa bore for receiving a reference electrode. Instead, the referenceelectrode 4515 is formed on or affixed to the bottom of central portion4520 and forms at least part of the flow chamber ceiling. For example, ametallization layer may be applied onto the bottom of central portion4520 before member 4510 is mounted onto the chip package.

FIGS. 47-48 show another example, which is a variant of the embodimentshown in FIGS. 43-44, but wherein the frame is manufactured as part ofthe flow cell rather attaching a flow port structure to a framepreviously attached to the chip surface. In designs of this type,assembly is somewhat more delicate since the wirebonds to the chip arenot protected by the epoxy encapsulating the chip. The success of thisdesign is dependent on the accurate placement and secure gluing of theintegrated “frame” to the surface of the chip. A counterpart embodimentto that of FIGS. 45-46, with the reference electrode 4910 on the ceilingof the flow chamber, and with the frame manufactured as part of the flowcell, is shown in FIGS. 49-50.

Yet another alternative for a fluidics assembly, as shown in FIGS.51-52, has a fluidics member 5110 raised by about 5.5 mm on stand-offs5120 from the top of the chip package 5130. This allows for an operatorto visually inspect the quality of the bonding between plastic piece5140 and chip surface and reinforce the bonding externally if necessary.

Some of the foregoing alternative embodiments also may be implemented ina hybrid plastic/PDMS configuration. For example, as shown in FIGS.53-54, a plastic part 5310 may make up the frame and flow chamber,resting on a PDMS “base” portion 5320. The plastic part 5310 may alsoprovides a region 5330 to the array, for expansion of the fluid flowfrom the inlet port; and the PDMS part may then include communicatingslits 5410, 5412 through which liquids are passed from the PDMS part toand from the flow chamber below.

The fluidic structure may also be made from glass as discussed above,such as photo-definable (PD) glass. Such a glass may have an enhancedetch rate in hydrofluoric acid once selectively exposed to UV light andfeatures may thereby be micromachined on the top-side and back-side,which when glued together can form a three-dimensional low aspect ratiofluidic cell.

An example is shown in FIG. 55. A first glass layer or sheet 5510 hasbeen patterned and etched to create nanoport fluidic holes 5522 and 5524on the top-side and fluid expansion channels 5526 and 5528 on theback-side. A second glass layer or sheet 5530 has been patterned andetched to provide downward fluid input/output channels 5532 and 5534, ofabout 300 μm height (the thickness of the layer). The bottom surface oflayer 5530 is thinned to the outside of channels 5532 and 5534, to allowthe layer 5530 to rest on the chip frame and protrusion area 5542 to beat an appropriate height to form the top of the flow channel. Two glasslayers, or wafers, and four lithography steps required. Both wafersshould be aligned and bonded (e.g., with an appropriate glue, not shown)such that the downward fluid input/output ports are aligned properlywith the fluid expansion channels. Alignment targets may be etched intothe glass to facilitate the alignment process.

Nanoports may be secured over the nanoport fluidic holes to facilitateconnection of input and output tubing.

A central bore 5550 may be etched through the glass layers for receivinga reference electrode, 5560. The electrode may be secured and sealed inplace with a silicone collar 5570 or like structure; or the electrodemay be equipped integrally with a suitable washer for effecting the samepurpose.

By using glass materials for the two-layer fluidic cell, the referenceelectrode may also be a conductive layer or pattern deposited on thebottom surface of the second glass layer (not shown). Or, as shown inFIG. 56, the protrusion region may be etched to form a permeable glassmembrane 5610 on the top of which is coated a silver (or other material)thin-film 5620 to form an integrated reference electrode. A hole 5630may be etched into the upper layer for accessing the electrode and ifthat hole is large enough, it can also serve as a reservoir for a silverchloride solution. Electrical connection to the thin-film silverelectrode may be made in any suitable way, such as by using a clip-onpushpin connector or alternatively wirebonded to the ceramic ISFETpackage.

Another alternative is to integrate the reference electrode to thesequencing chip/flow cell by using a metalized surface on the ceiling ofthe flow chamber—i.e., on the underside of the member forming theceiling of the fluidic cell. An electrical connection to the metalizedsurface may be made in any of a variety of ways, including, but notlimited to, by means of applying a conductive epoxy to the ceramicpackage seal ring that, in turn, may be electrically connected through avia in the ceramic substrate to a spare pin at the bottom of the chippackage. Doing this would allow system-level control of the referencepotential in the fluid cell by means of inputs through the chip socketmount to the chip's control electronics.

By contrast, an externally inserted electrode requires extra fluidtubing to the inlet port, which requires additional fluid flow betweencycles.

Ceramic pin grid array (PGA) packaging may be used for the ISFET array,allowing customized electrical connections between various surfaces onthe front face with pins on the back.

The flow cell can be thought of as a “lid” to the ISFET chip and itsPGA. The flow cell, as stated elsewhere, may be fabricated of manydifferent materials. Injection molded polycarbonate appears to be quitesuitable. A conductive metal (e.g., gold) may be deposited using anadhesion layer (e.g., chrome) to the underside of the glow cell roof(the ceiling of the flow chamber). Appropriate low-temperature thin-filmdeposition techniques preferably are employed in the deposition of themetal reference electrode due to the materials (e.g., polycarbonate) andlarge step coverage topography at the bottom-side of the fluidic cell(i.e., the frame surround of ISFET array). One possible approach wouldbe to use electron-beam evaporation in a planetary system.

The active electrode area is confined to the central flow chamber insidethe frame surround of the ISFET array, as that is the only metalizedsurface that would be in contact with the ionic fluid during sequencing.

Once assembly is complete—conductive epoxy (e.g., Epo-Tek H20E orsimilar) may be dispensed on the seal ring with the flow cell aligned,placed, pressed and cured—the ISFET flow cell is ready for operationwith the reference potential being applied to the assigned pin of thepackage.

The resulting fluidic system connections to the ISFET device thusincorporate shortened input and output fluidic lines, which isdesirable.

Still another example embodiment for a fluidic assembly is shown inFIGS. 57-58. This design is limited to a plastic piece 5710 whichincorporates the frame and is attached directly to the chip surface, andto a second piece 5720 which is used to connect tubing from the fluidicsystem and similarly to the PDMS piece discussed above, distributes theliquids from the small bore tube to a wide flat slit. The two pieces areglued together and multiple (e.g., three) alignment markers (not shown)may be used to precisely align the two pieces during the gluing process.A hole may be provided in the bottom plate and the hole used to fill thecavity with an epoxy (for example) to protect the wirebonds to the chipand to fill in any potential gaps in the frame/chip contact. In theillustrated example, the reference electrode is external to the flowcell (downstream in the exhaust stream, through the outlet port—seebelow), though other configurations of reference electrode may, ofcourse, be used.

Still further examples of flow cell structures are shown in FIGS. 59-60.FIG. 59A comprises eight views (A-H) of an injection molded bottomlayer, or plate, 5910, for a flow cell fluidics interface, while FIG.59B comprises seven views (A-G) of a mating, injection molded top plate,or layer, 5950. The bottom of plate 5910 has a downwardly depending rim5912 configured and arranged to enclose the sensor chip and an upwardlyextending rim 5914 for mating with the top plate 5610 along its outeredge. To form two fluid chambers (an inlet chamber and an outletchamber) between them. A stepped, downwardly depending portion 5960 oftop plate 5950, separates the input chamber from the output chamber. Aninlet tube 5970 and an outlet tube 5980 are integrally molded with therest of top plate 5950. From inlet tube 5970, which empties at the smallend of the inlet chamber formed by a depression 5920 in the top of plate5910, to the outlet edge of inlet chamber fans out to direct fluidacross the whole array.

Whether glass or plastic or other material is used to form the flowcell, it may be desirable, especially with larger arrays, to include inthe inlet chamber of the flow cell, between the inlet conduit and thefront edge of the array, not just a gradually expanding (fanning out)space, but also some structure to facilitate the flow across the arraybeing suitably laminar. Using the bottom layer 5990 of an injectionmolded flow cell as an example, one example type of structure for thispurpose, shown in FIG. 59C, is a tree structure 5992 of channels fromthe inlet location of the flow cell to the front edge of the microwellarray or sensor array, which should be understood to be under the outletside of the structure, at 5994.

The above-described systems for sequencing typically utilize a laminarfluid flow system to sequence a biological polymer. In part, the fluidflow system preferably includes a flow chamber formed by the sensor chipand a single piece, injection molded member comprising inlet and outletports and mountable over the chip to establish the flow chamber. Thesurface of such member interior to the chamber is preferably formed tofacilitate a desired expedient fluid flow, as described herein.

In some embodiments, the invention encompasses an apparatus fordetection of ion pulses comprising a laminar fluid flow system.Preferably, the apparatus is used for sequencing a plurality of nucleicacid templates, wherein the nucleic acid templates are optionallydeposited on an array.

The apparatus typically includes a fluidics assembly comprising a membercomprising one or more apertures for non-mechanically directing a fluidto flow to an array of at least 100K, 500K, or 1M microfluidic reactionchambers such that the fluid reaches all of the microfluidic reactionchambers at the same time or substantially the same time. Typically, thefluid flow is parallel to the sensor surface. Typically, the assemblyhas a Reynolds number of less than 1000, 500, 200, 100, 50, 20, or 10.Preferably, the member further comprises a first aperture for directingfluid towards the sensor array and a second aperture for directing fluidaway from the sensor array.

In some embodiments, the invention encompasses a method for directing afluid to a sensor array comprising: providing a fluidics assemblycomprising an aperture fluidly coupling a fluid source to the sensorarray; and non-mechanically directing a fluid to the sensor array. By“non-mechanically” it is meant that the fluid is moved under pressurefrom a gaseous pressure source, as opposed to a mechanical pump.

In some embodiments, the invention encompasses an array of wells, eachof which is coupled to a lid having an inlet port and an outlet port anda fluid delivery system for delivering and removing fluid from saidinlet and outlet ports non-mechanically.

In some embodiments, the invention encompasses a method for sequencing abiological polymer utilizing the above-described apparatus, comprising:directing a fluid comprising a monomer to an array of reaction chamberswherein the fluid has a fluid flow Reynolds number of at most 2000,1000, 200, 100, 50, or 20. The method may optionally further comprisedetecting an ion pulse from each said reaction chamber. The ion pulse istypically detected by ion diffusion to the sensor surface. There arevarious other ways of providing a fluidics assembly for delivering anappropriate fluid flow across the microwell and sensor array assembly,and the forgoing examples are thus not intended to be exhaustive.

Commercial flow-type fluidic electrodes, such as silver chlorideproton-permeable electrodes, may be inserted in series in a fluidic lineand are generally designed to provide a stable electrical potentialalong the fluidic line for various electrochemical purposes. In theabove-discussed system, however, such a potential must be maintained atthe fluidic volume in contact with the microwell ISFET chip. Withconventional silver chloride electrodes, it has been found difficult,due to an electrically long fluidic path between the chip surface andthe electrode (through small channels in the flow cell), to achieve astable potential. This led to reception of noise in the chip'selectronics. Additionally, the large volume within the flow cavity ofthe electrode tended to trap and accumulate gas bubbles that degradedthe electrical connection to the fluid. With reference to FIG. 60, asolution to this problem has been found in the use of a stainless steelcapillary tube electrode 6010, directly connected to the chip's flowcell outlet port 6020 and connected to a voltage source (not shown)through a shielded cable 6030. The metal capillary tube 6010 has a smallinner diameter (e.g., on the order of 0.01″) that does not trap gas toany appreciable degree and effectively transports fluid and gas likeother microfluidic tubing. Also, because the capillary tube can bedirectly inserted into the flow cell port 6020, it close to the chipsurface, reducing possible electrical losses through the fluid. Thelarge inner surface area of the capillary tube (typically about 2″ long)may also contribute to its high performance. The stainless steelconstruction is highly chemically resistant, and should not be subjectto electrochemical effects due to the very low electrical current use inthe system (<1 μA). A fluidic fitting 6040 is attached to the end of thecapillary that is not in the flow cell port, for connection to tubing tothe fluid delivery and removal subsystem.

A complete system for using the sensor array will include suitable fluidsources, valving and a controller for operating the valving to lowreagents and washes over the microarray or sensor array, depending onthe application. These elements are readily assembled from off-the-shelfcomponents, with and the controller may readily be programmed to performa desired experiment.

It should be understood that the readout at the chemFET may be currentor voltage (and change thereof) and that any particular reference toeither readout is intended for simplicity and not to the exclusion ofthe other readout. Therefore any reference in the following text toeither current or voltage detection at the chemFET should be understoodto contemplate and apply equally to the other readout as well. Inimportant embodiments, the readout reflects a rapid, transient change inconcentration of an analyte. The concentration of more than one analytemay be detected at different times. Such measurements are to becontrasted with prior art methods which focused on steady stateconcentration measurements.

As already discussed, the apparatus and systems of the invention can beused to detect and/or monitor interactions between various entities.These interactions include biological and chemical reactions and mayinvolve enzymatic reactions and/or non-enzymatic interactions such asbut not limited to binding events. As an example, the inventioncontemplates monitoring enzymatic reactions in which substrates and/orreagents are consumed and/or reaction intermediates, byproducts and/orproducts are generated. An example of a reaction that can be monitoredaccording to the invention is a nucleic acid synthesis method such asone that provides information regarding nucleic acid sequence. Thisreaction will be discussed in greater detail herein.

In the context of a sequencing reaction, the apparatus and systemprovided herein is able to detect nucleotide incorporation based onchanges in the chemFET current and/or voltage, as those latterparameters are interrelated. Current changes may be the result of one ormore of the following events either singly or some combination thereof:generation of PPi, generation of Pi (e.g., in the presence ofpyrophosphatase), generation of hydrogen (and concomitant changes in pHfor example in the presence of low strength buffer), reducedconcentration of unincorporated dNTP at the chemFET surface, delayedarrival of unincorporated dNTP at the chemFET surface, and the like. Themethods described herein are able to detect changes in analyteconcentration at the chemFET surface, and such changes may result fromone or more of the afore-mentioned events. The invention contemplatesthe use of a chemFET such as an ISFET in the sequencing methodsdescribed herein, even if the readout is independent of (or insensitiveto) pH. In other words, the invention contemplates the use of an ISFETfor the detection of analytes such as PPi and unincorporatednucleotides. The methods provided herein in regards to sequencing can becontrasted to those in the literature including Pourmand et al. PNAS2006 103(17):6466-6470. As discussed herein, the invention contemplatesmethods for determining the nucleotide sequence (i.e., the “sequence”)of a nucleic acid. Such methods involve the synthesis of a new nucleicacid (primed by a pre-existing nucleic acid, as will be appreciated bythose of ordinary skill), based on the sequence of a template nucleicacid. That is, the sequence of the newly synthesized nucleic acid iscomplimentary to the sequence of the template nucleic acid and thereforeknowledge of sequence of the newly synthesized nucleic acid yieldsinformation about the sequence of the template nucleic acid. Knowledgeof the sequence of the newly synthesized nucleic acid is derived bydetermining whether a known nucleotide has been incorporated into thenewly synthesized nucleic acid and, if so, how many of such knownnucleotides have been incorporated. Nucleotide incorporation can bemonitored in a number of ways, including the production of products suchas PPi, Pi and/or H.

FIG. 61 illustrates the production/generation of PPi resulting from theincorporation of a nucleotide in a newly synthesized nucleic acidstrand. PPi generated as a reaction byproduct of nucleotideincorporation in a nucleic acid strand can be detected directly even inthe absence of a PPi receptor (such as those provided in FIG. 11B1-3)and in the absence of a detectable pH change (e.g., as may occur in thepresence of a strong buffer, as defined herein). The simple presence ofPPi is sufficient, in some instances, to cause an electrical change inthe chemFET surface, thereby resulting in a current change. The currentchange may result from PPi generation alone or in combination with otherevents such as those described above.

FIG. 8A shows the calculated concentrations of substrates and productsof a nucleotide incorporation reaction. These calculations are based ona model in which a plurality of nucleic acids are synchronouslysynthesized from a plurality of identical template nucleic acids boundto a bead situated in a well, as described herein. The model assumesthat the wells contain template nucleic acids hybridized to primernucleic acids, polymerases, and co-factors needed for nucleotideincorporation, except for nucleotides. The incorporation reaction isinitiated by introducing (through flow) a population of known, identicalnucleotide triphosphates into the well. The model indicates that thefirst analyte detected by the chemFET surface is the newly generatedPPi, and that at some time point thereafter unincorporated nucleotidetriphosphates are generated. Each of these analyte populations will giverise to a current readout at the chemFET. FIG. 8B shows the voltagepulses and/or peaks (referred to herein interchangeably) that correspondto changes in the concentration of PPi and unincorporated nucleotides.The time of the first observable pulse is referred to herein as t₀ andthe time of the second observable pulse is referred to herein as t₁. Thevarious overlayed traces reflect the incorporation of none, one or moreidentical nucleotides in a given reaction. As can be seen in FIG. 8B,the difference in time between t₁ and t₀ (i.e., t₁−t₀) increases withincreasing nucleotide incorporation. As a result, the time differencebetween the PPi pulse and the unincorporated nucleotide triphosphatepulse may be used as a measure of how many nucleotide triphosphates havebeen incorporated in any given step. This will typically be used todetermine how many of a known single type of dNTP is incorporated.However it may also be used to determine the number of dNTP incorporatedregardless of the identity of the dNTP according to other aspects of theinvention.

Thus, some embodiments the invention contemplate that upon introductionof nucleotides that are complementary to the next position to besequenced on the template nucleic acid, such nucleotides will beeffectively consumed yielding PPi which is then detected at the chemFET.Only once the synthesis reactions have gone to completion (i.e., theappropriate number of nucleotides have been incorporated into all newlysynthesized nucleic acids) is the population of unincorporatednucleotides able to diffuse to and be detected by the chemFET surface.The delay in the detection of the unincorporated nucleotides isindicative of how many such nucleotides have been incorporated. Thesedelays are modeled in FIG. 8B. If the nucleotides introduced into thewells are not complimentary to the next position to be sequenced on thetemplate nucleic acid, then no incorporation should occur. Accordingly,the first analyte detected at the surface of the chemFET will be thepopulation of unincorporated nucleotides since no PPi would begenerated. Thus, detection of a single ionic pulse by the chemFET (whichis translated into a single voltage pulse) indicates that no nucleotideincorporation has occurred, detection of two, temporally separated,ionic pulses (which are translated into two voltage pulses) indicatesthat nucleotide incorporation has occurred, and the time differencebetween the latter two pulses indicates the number of nucleotides thathave been incorporated.

The incorporation of a dNTP into the nucleic acid strand releases PPiwhich can then be hydrolyzed to two orthophosphates (Pi) and onehydrogen ion. The generation of the hydrogen ion therefore can bedetected as an indicator of nucleotide incorporation. Alternatively, Pimay be detected directly or indirectly. Any and all of these events (andmore as described herein) may be detected at the chemFET thereby causinga current change that correlates with nucleotide incorporation.

The limit of this dual pulse detection occurs when the two pulses cannotbe resolved in time (i.e., the two peaks may merge into one, where t₀and t₁ peaks are not distinguishable). This limit, for the caseN_(poly)=0 vs N_(poly)=1 is given by the following: Consider a firstpeak N_(poly)=0 which has a width=W₀ and standard deviation σ_(W) ₀ anda second peak N_(poly)=1 which has a width=W₁ and standard deviationσ_(W) ₁ . For a P_(e) of 1% [where P_(e)=erfc(SNR/2*(sqr(2)))], thesignal-to-noise ratio (SNR)=5.15. Therefore the limit of detection isthe difference in peak widths, ΔW, is given by ΔW=(W₁−W₀)>5.15*σ_(W) ₀ .Similar calculations may be done for other SNRs.

In certain embodiments the chemFET (or ISFET) readouts are independentof (or insensitive to) changes in the concentration of Pi and/or H⁺. Inthese embodiments, the rate of PPi hydrolysis to Pi with the concomitantrelease of H⁺ is reduced, sometimes significantly. The PPi to Piconversion may be catalyzed by an enzyme such as a pyrophosphataseand/or by certain ions. In the absence of the both (and in some cases,either) the enzyme and the particular ion, the rate of conversion issufficiently slow to be negligible. (See for example J. Chem, Soc.Farady Trans. 2007, 93:4295 which reports the rate constant for theconversion in the absence of enzyme or ion to be on the order of about3⁻¹ years⁻¹, as compared to Biochemistry, 2002, 41:12025 which reportsthe rate in the presence of enzyme and Mg²⁺ to be on the order of about10³ s⁻¹.) A pH insensitive environment may be one that is bufferedsufficiently to obscure any changes in pH resulting from release ofhydrogen. The reaction may be pH insensitive or independent as a resultof there being few if any hydrogen ions released as a result of thereaction.

The nucleic acid being sequenced is referred to herein as the targetnucleic acid. Target nucleic acids include but are not limited to DNAsuch as but not limited to genomic DNA, mitochondrial DNA, cDNA and thelike, and RNA such as but not limited to mRNA, miRNA, and the like. Thenucleic acid may be from any source including naturally occurringsources or synthetic sources. The nucleic acids may be PCR products,cosmids, plasmids, naturally occurring or synthetic libraries, and thelike. The invention is not intended to be limited in this regard. Themethods provided herein can be used to sequence nucleic acids of anylength. To be clear, the Examples provide a proof of principledemonstration of the sequencing of four templates of known sequence.This artificial model is intended to show that embodiments of theapparatuses and systems described herein are able to readout nucleotideincorporation that correlates to the known sequence of the templates.This is not intended to represent typical use of the method or system inthe field. The following is a brief description of these methods.

Target nucleic acids are prepared using any manner known in the art. Asan example, genomic DNA may be harvested from a sample according totechniques known in the art (see for example Sambrook et al.“Maniatis”). Following harvest, the DNA may be fragmented to yieldnucleic acids of smaller length. The resulting fragments may be on theorder of hundreds, thousands, or tens of thousands nucleotides inlength. In some embodiments, the fragments are 200-1000 base pairs insize, or 300-800 base pairs in size, about 200, about 300, about 400,about 500, about 600, about 700, about 800, about 900, or about 1000base pairs in length, although they are not so limited. Nucleic acidsmay be fragmented by any means including but not limited to mechanical,enzymatic or chemical means. Examples include shearing, sonication,nebulization, endonuclease (e.g., DNase I) digestion, amplification suchas PCR amplification, or any other technique known in the art to producenucleic acid fragments, preferably of a desired length. As used herein,fragmentation also embraces the use of amplification to generate apopulation of smaller sized fragments of the target nucleic acid. Thatis, the target nucleic acids may be melted and then annealed to two (andpreferably more) amplification primers and then amplified using forexample a thermostable polymerase (such as Taq). An example is amassively parallel PCR-based amplification. Fragmentation can befollowed by size selection techniques to enrich or isolate fragments ofa particular length or size. Such techniques are also known in the artand include but are not limited to gel electrophoresis or SPRI.

Alternatively, target nucleic acids that are already of sufficient smallsize (or length) may be used. Such target nucleic acids include thosederived from an exon enrichment process. Thus, rather than fragmenting(randomly or non-randomly) longer target nucleic acids, the targets maybe nucleic acids that naturally exist or can be isolated in shorter,useable lengths such as mRNAs, cDNAs, exons, PCR products (as describedabove), and the like. See Albert et al. Nature Methods 20074(11):903-905 (microarray hybridization of exons and locus-specificregions), Porreca et al. Nature Methods 2007 4(11):931-936, and Okou etal. Nature Methods 2007 4(11):907-909 for methods of isolating and/orenriching sequences such as exons prior to sequencing.

In some embodiments, the size selected target nucleic acids are ligatedto adaptor sequences on both the 5′ and 3′ ends. These adaptor sequencescomprise sequences complementary to amplification primer sequences, tobe used in amplifying the target nucleic acids. One adaptor sequence mayalso comprise a sequence complementary to the sequencing primer. Theopposite adaptor sequence may comprise a moiety that facilitates bindingof the nucleic acid to a solid support such as but not limited to abead. An example of such a moiety is a biotin molecule (or a doublebiotin moiety, as described by Diehl et al. Nature Methods, 2006,3(7):551-559) and such a labeled nucleic acid can therefore be bound toa solid support having avidin or streptavidin groups. Another moietythat can be used is the NHS-ester and amine affinity pair. It is to beunderstood that the invention is not limited in this regard and one ofordinary skill is able to substitute these affinity pairs with otherbinding pairs. The resulting nucleic acid is referred to herein as atemplate nucleic acid. The template nucleic acid comprises at least thetarget nucleic acid and usually comprises nucleotide sequences inaddition to the target at both the 5′ and 3′ ends.

In some embodiments, the template nucleic acid is able to self-annealthereby creating a 3′ end from which to incorporate nucleotidetriphosphates. In such instances, there is no need for a separatesequencing primer since the template acts as both template and primer.See Eriksson et al. Electrophoresis 2004 25:20-27 for a discussion ofthe use of self-annealing template in a pyrosequencing reaction.

In still other embodiments, the template is used in a double stranded(or partially double stranded) from which is nicked preferably within anadaptor sequence (at the free end of the bead bound template). Theopening in the double stranded template allows a polymerase such as DNApolymerase to enter at the nicked site and begin nucleotideincorporation. These openings can be introduced into the template in acontrolled manner particularly since the sequence acted upon by DNAnickase (i.e., the nicking enzyme in this instance) is know (i.e.,5′GAGTC3′). See Zyrina et al. for a discussion of these consensusnicking target sequences.

In some instances a spacer is used to distance the template nucleic acid(and in particular the target nucleic acid sequence comprised therein)from a solid support such as a bead. This facilitates sequencing of theend of the target closest to the bead, for instance. Examples ofsuitable linkers are known in the art (see Diehl et al. Nature Methods,2006, 3(7):551-559) and include but are not limited to carbon-carbonlinkers such as but not limited to iSp18.

The solid support to which the template nucleic acids are bound isreferred to herein as the “capture solid support”. If the solid supportis a bead, then such bead is referred to herein as a “capture bead”. Thebeads can be made of any material including but not limited tocellulose, cellulose derivatives, gelatin, acrylic resins, glass, silicagels, polyvinyl pyrrolidine (PVP), co-polymers of vinyl and acrylamide,polystyrene, polystyrene cross-linked with divinylbenzene or the like(see, Merrifield Biochemistry 1964, 3, 1385-1390), polyacrylamides,latex gels, dextran, crosslinked dextrans (e.g., Sephadex™), rubber,silicon, plastics, nitrocellulose, natural sponges, metal, and agarosegel (Sepharose™). In one embodiment, the beads are streptavidin-coatedbeads. The bead diameter will depend on the density of the chemFET andmicrowell array used with larger arrays (and thus smaller sized wells)requiring smaller beads. Generally the bead size may be about 1-10 μM,and more preferably 2-6 μM. In some embodiments, the beads are about5.91 μM while in other embodiments the beads are about 2.8 μM. In stillother embodiments, the beads are about 1.5 μm, or about 1 μm indiameter. It is to be understood that the beads may or may not beperfectly spherical in shape. It is also to be understood that otherbeads may be used and other mechanisms for attaching the nucleic acid tothe beads may be used. In some instances the capture beads (i.e., thebeads on which the sequencing reaction occurs) are the same as thetemplate preparation beads including the amplification beads.

Important aspects of the invention contemplate sequencing a plurality ofdifferent template nucleic acids simultaneously. This may beaccomplished using the sensor arrays described herein. In oneembodiment, the sensor arrays are overlayed (and/or integral with) anarray of microwells (or reaction chambers or wells, as those terms areused interchangeably herein), with the proviso that there be at leastone sensor per microwell. Present in a plurality of microwells is apopulation of identical copies of a template nucleic acid. There is norequirement that any two microwells carry identical template nucleicacids, although in some instances such templates may share overlappingsequence. Thus, each microwell comprises a plurality of identical copiesof a template nucleic acid, and the templates between microwells may bedifferent.

The microwells may vary in size between arrays. The microwell size maybe described in terms of cross section. The cross section may refer to a“slice” parallel to the depth (or height) of the well, or it may be aslice perpendicular to the depth (or height) of the well.

The size of these microwells may be described in terms of a width (ordiameter) to height ratio. In some embodiments, this ratio is 1:1 to1:1.5. The bead to well size (e.g., the bead diameter to well width,diameter, or height) is preferably in the range of 0.6-0.8.

The microwells may be square in cross-section, but they are not solimited. The dimensions at the bottom of a microwell (i.e., in a crosssection that is perpendicular to the depth of the well) may be 1.5 μm by1.5 μm, or it may be 1.5 μm by 2 μm. Various diameters are shown in theExamples and include but are not limited to diameters at or about 100μm, 95 μm, 90 μm, 85 μm, 80 μm, 75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50μm, 45 μm, 40 μm, 35 μm, 30 μm, 25 μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm,7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm, 1 μm or less. In some particularembodiments, the diameters may be at or about 44 μm, 32 μm, 8 μm, 4 μm,or 1.5 μm. Various heights are shown in the Examples and include but arenot limited to heights at or about 100 μm, 95 μm, 90 μm, 85 μm, 80 μm,75 μm, 70 μm, 65 μm, 60 μm, 55 μm, 50 μm, 45 μm, 40 μm, 35 μm, 30 μm, 25μm, 20 μm, 15 μm, 10 μm, 9 μm, 8 μm, 7 μm, 6 μm, 5 μm, 4 μm, 3 μm, 2 μm,1 μm or less. In some particular embodiments, the heights may be at orabout 55 μm, 48 μm, 32 μm, 12 μm, 8 μM, 6 μm, 4 μm, 2.25 μm, 1.5 μm, orless. Various embodiments of the invention contemplate the combinationof any of these diameters with any of these heights. In still otherembodiments, the reaction well dimensions may be (diameter in μm byheight in μm) 44 by 55, 32 by 32, 32 by 48, 8 by 8, 8 by 12, 4 by 4, 4by 6, 1.5 by 1.5, or 1.5 by 2.25.

The reaction well volume may range (between arrays, and preferably notwithin a single array) based on the well dimensions. This volume may beat or about 100 picoliter (pL), 90, 80, 70, 60, 50, 40, 30, 20, 10, orfewer pL. In important embodiments, the well volume is less than 1 pL,including equal to or less than 0.5 pL, equal to or less than 0.1 pL,equal to or less than 0.05 pL, equal to or less than 0.01 pL, equal toor less than 0.005 pL, or equal to or less than 0.001 pL. The volume maybe 0.001 to 0.9 pL, 0.001 to 0.5 pL, 0.001 to 0.1 pL, 0.001 to 0.05 pL,or 0.005 to 0.05 pL. In particular embodiments, the well volume is 75pL, 34 pL, 23 pL, 0.54 pL, 0.36 pL, 0.07 pL, 0.045 pL, 0.0024 pL, or0.004 pL. The plurality of templates in each microwell may be introducedinto the microwells (e.g., via a nucleic acid loaded bead), or it may begenerated in the microwell itself. A plurality is defined herein as atleast two, and in the context of template nucleic acids in a microwellor on a nucleic acid loaded bead includes tens, hundreds, thousands, tenthousands, hundred thousands, millions, or more copies of the templatenucleic acid. The limit on the number of copies will depend on a numberof variables including the number of binding sites for template nucleicacids (e.g., on the beads or on the walls of the microwells), the sizeof the beads, the length of the template nucleic acid, the extent of theamplification reaction used to generate the plurality, and the like. Itis generally preferred to have as many copies of a given template perwell in order to increase signal to noise ratio as much as possible.Amplification and conjugation of nucleic acids to solid supports such asbeads may be accomplished in a number of ways, including but not limitedto emulsion PCR (i.e., water in oil emulsion amplification) as describedby Margulies et al. Nature 2005 437(15):376-380 and accompanyingsupplemental materials. In some embodiments, the amplification is arepresentative amplification. A representative amplification is anamplification that does not alter the relative representation of anynucleic acid species. The wells generally also include sequencingprimers, polymerases and other substrates or catalysts necessary for thesynthesis reaction.

The degree of saturation of any capture (i.e., sequencing) bead withtemplate nucleic acid to be sequenced may not be 100%. In someembodiments, a saturation level of 10%-100% exists. As used herein, thedegree of saturation of a capture bead with a template refers to theproportion of sites on the bead that are conjugated to template. In someinstances this may be at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or it may be100%.

It will be understood that the amount of sequencing primers andpolymerases may be saturating, above saturating level, or in someinstances below saturating levels. As used herein, a saturating level ofa sequencing primer or a polymerase is a level at which every templatenucleic acid is hybridized to a sequencing primer or bound by apolymerase, respectively. Thus the saturating amount is the number ofpolymerases or primers that is equal to the number of templates on asingle bead. In some embodiments, the level is at greater than this,including at least 2 fold, 3 fold, 4 fold, 5 fold, 10 fold, or more overthe level of the template nucleic acid. In other embodiments, the numberof polymerases and/or primers may be 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, or up to 100% of the number of templates on a single bead in asingle well.

Thus, for example, before and/or while in the wells, the templatenucleic acids are incubated with a sequencing primer that binds to itscomplementary sequence located on the 3′ end of the template nucleicacid (i.e., either in the amplification primer sequence or in anotheradaptor sequence ligated to the 3′ end of the target nucleic acid) andwith a polymerase for a time and under conditions that promotehybridization of the primer to its complementary sequence and thatpromote binding of the polymerase to the template nucleic acid. Theprimer can be of virtually any sequence provided it is long enough to beunique. The hybridization conditions are such that the primer willhybridize to only its true complement on the 3′ end of the template.Suitable conditions are disclosed in Margulies et al. Nature 2005437(15):376-380 and accompanying supplemental materials.

As described herein, the template nucleic acids may be engineered suchthat different templates have identical 5′ ends and identical 3′ ends.In some embodiments, however, the invention contemplates the use of aplurality of template populations, wherein each member of a givenplurality shares the same 3′ end but different template populationsdiffer from each other based on their 3′ end sequences. As an example,the invention contemplates in some instances sequencing nucleic acidsfrom more than one subject or source. Nucleic acids from first sourcemay have a first 3′ sequence, nucleic acids from a second source mayhave a second 3′ sequence, and so on, provided that the first and second3′ sequences are different. In this respect, the 3′ end, which istypically a unique sequence, can be used as a barcode or identifier tolabel (or identify) the source of the particular nucleic acid in a givenwell. Reference can be made to Meyer et al. Nucleic Acids Research 200735(15):e97 for a discussion of labeling nucleic acid with barcodesfollowed by sequencing. In some instances, the sequencing primers (ifused) may be hybridized (or annealed, as the terms are usedinterchangeably herein) to the templates prior to loading (orintroducing) the beads to the wells or after such loading.

The 5′ and 3′ ends on every individual template however are preferablydifferent in sequence. In particular, the templates share identicalprimer binding sequences. This facilitates the use of an identicalprimer across microwells and also ensures that a similar (or identical)degree of primer hybridization occurs across microwells. Once annealedto complementary primers such as sequencing primers, the templates arein a complex referred to herein as a template/primer hybrid. In thishybrid, one region of the template is double stranded (i.e., where it isbound to its complementary primer) and one region is single stranded. Itis this single stranded region that acts as the template for theincorporation of nucleotides to the end of the primer and thus it isalso this single stranded region which is ultimately sequenced accordingto the invention.

Suitable polymerases include but are not limited to DNA polymerase, RNApolymerase, or a subunit thereof, provided it is capable of synthesizinga new nucleic acid strand based on the template and starting from thehybridized primer. An example of a suitable polymerase subunit is theexo− version of the Klenow fragment of E. coli DNA polymerase I whichlacks 3′ to 5′ exonuclease activity. Other polymerases include T4 exo−,Therminator, and Bst polymerases. The polymerase may be free in solution(and may be present in wash and dNTP solutions) or it may be bound forexample to the beads (or corresponding solid support) or to the walls ofthe chemFET but preferably not to the ISFET surface itself. In anotheraspect, the invention provides a method for sequencing a nucleic acidcomprising disposing a plurality of template nucleic acids into aplurality of reaction chambers and each reaction chamber is in contactwith a chemFET. Sequencing includes synthesizing a new nucleic acidstrand by incorporating one or more known nucleotide triphosphatessequentially at the 3′ end of the sequencing primer, and detectingincorporation of the one or more known nucleotide triphosphates. Theincorporation is carried out by a DNA polymerase. In some embodiments,the polymerase has high affinity for the template, and has aprocessivity of 100 bp, 250 bp, 500 bp, 750 bp or even 1000 bp. In someembodiments, the polymerase has high activity and a high catalytic ratein high pH conditions. In some embodiments, the pH of the reactionmixture is about 7 to 10, and preferably about 9. In some embodiments,the enzyme has high activity and a high catalytic rate in lowconcentrations of dNTPs. In some embodiments the dNTP concentration is50 μM, 40 μM, 30 μM, 20 μM, 10 μM, 5 μM, and preferably 20 μM or less.In some embodiments the enzyme has high activity a catalytic rate wherethe reaction mixture is of low ionic strength. In some embodiments theconcentration of MgCl₂ or MnCl² is 100 μM or less. In some embodimentsthe concentration of Tris is 0.5 mM or less.

Various aspects of the invention relate to nucleotide incorporation intoa pre-existing nucleic acid (such as for example a sequencing primer) inthe presence of low ionic strength solutions or environments. As usedherein, a low ionic strength solution is a solution that has an ionconcentration equal to or less than 3 mM total ion concentration. Anexample of a low ionic strength solution is a 0.5 mM Tris-HCl solutionhaving less than 100 μM MgCl₂. The MgCl₂ concentration may be equal toor less than 90 μM, equal to or less than 80 μM, equal to or less than70 μM, equal to or less than 60 μM, equal to or less than 50 μM, equalto or less than 40 μM, equal to or less than 30 μM, equal to or lessthan 20 μM, equal to or less than 10 μM, or less. The polymerase in someembodiments therefore must be capable of functioning at low ionicstrength. As described in the Examples, polymerases preferably will beable to recognize and bind to the primer/template hybrid, havesufficient affinity for the hybrid resulting for example in increasedprocessivity of the polymerase, and extend the primer, in low ionicstrength conditions. Example 2 demonstrates these various properties forT4 exo−, Therminator, Bst, and Klenow exo− polymerases. The Examplefurther demonstrates that these polymerases are able to incorporatenucleotides into and thereby extend a primer in the context of a siliconwell in contact with a chemFET.

In some embodiments, the polymerase must also be capable ofincorporating nucleotides in low concentrations of dNTPs. In someembodiments, a low concentration of dNTPs is a concentration that isless 20 μM total dNTP. It will be understood by those of ordinary skillin the art that this concentration relates to the concentration of eachdNTP added to the reaction wells, in those embodiments contemplatingsequential rather than concurrent introduction of dNTP to a reactionwell. In other embodiments, the dNTP concentration (either per dNTP orin some instances total dNTP present in a reaction well at any timeduring a reaction) is equal to or less than 19 μM, equal to or less than18 μM, equal to or less than 17 μM, equal to or less than 16 μM, equalto or less than 15 μM, equal to or less than 14 μM, equal to or lessthan 13 μM, equal to or less than 12 μM, equal to or less than 11 μM,equal to or less than 10 μM, equal to or less than 9 μM, equal to orless than 8 μM, equal to or less than 7 μM, equal to or less than 6 μM,equal to or less than 5 μM, equal to or less than 4 μM, equal to or lessthan 3 μM, equal to or less than 2 μM, equal to or less than 1 μM, orless. In some embodiments, the dNTP concentration is between 0-20 μM,between 1-19 μM, or between 5-15 μM.

Some embodiments of the invention require that the polymerase havesufficient processivity. As used herein, processivity is the ability ofa polymerase to remain bound to a single primer/template hybrid. As usedherein, it is measured by the number of nucleotides that a polymeraseincorporates into a nucleic acid (such as a sequencing primer) prior todisassociation of the polymerase from the primer/template hybrid. Insome embodiments, the polymerase has a processivity of at least 100nucleotides, although in other embodiments it has a processivity of atleast 200 nucleotides, at least 300 nucleotides, at least 400nucleotides, or at least 500 nucleotides. It will be understood those ofordinary skill in the art that the higher the processivity of thepolymerase, the more nucleotides that can be incorporated prior todisassociation, and therefore the longer the nucleic acid that can besequenced. In other words, polymerases having low processivity (e.g., apolymerase that dissociates from the hybrid after five incorporationswill only provide sequence of 5 nucleotides in length, according to someaspects of the invention.

Still other aspects and embodiments of the invention contemplateperforming nucleotide incorporation at a high pH (i.e., a pH that isgreater than 7). Thus, some reactions are carried out at a pH equal toor greater than 7.5, equal to or greater than 8, equal to or greaterthan 8.5, equal to or greater than 9, equal to or greater than 9.5,equal to or greater than 10, or equal to or greater than 11. Thepolymerase may be one that incorporates nucleotides into a nucleic acid(such as a sequencing primer) at a pH of 7-11, 7.5-10.5, 8-10, 8.5-9.5,or at about 9.

It will be understood based on the teachings herein that some aspectsand embodiments of the invention will employ polymerases demonstratinghigh activity in low ionic strength solutions, and/or low dNTPconcentrations, and/or high pH. Suitable rates of incorporation willvary depending on the embodiment. In some embodiments, nucleotideincorporation occurs at a rate faster than the diffusion of reagentswithin a reaction chamber. For example, in one embodiment, nucleotideincorporation occurs at a rate that exceeds the rate of diffusion ofdNTP in the reaction well and preferably of dNTP towards the bottom orthe reaction well (i.e., to the sensor). In these instances, the newlyintroduced dNTP do not diffuse towards the sensor until theincorporation reactions are complete. At that time, the remaining dNTP(i.e., the unincorporated dNTP) diffuse past the hybrids (or the beads,as the case may be) and towards the sensor bottom. In these instances,until the incorporation reactions are complete, the dNTP diffusing inthe direction of the bead and the well bottom are used up before evenreaching the well bottom.

The rate at which a polymerase incorporates nucleotides will varydepending on the particular application, although generally faster ratesof incorporation are preferable. The rate of incorporation of a singlepolymerase to a single nucleic acid (such as a sequencing primer) insome instances is faster than the rate of diffusion of theunincorporated nucleotide triphosphates in the well, and particularlytheir diffusion towards the bottom of the well. The rate of “sequencing”will depend on the number of arrays on chip, the size of the wells, thetemperature and conditions at which the reactions are run, etc. Asdiscussed elsewhere herein, the diffusion rate can also be manipulatedby effectively impeding the movement of unincorporated nucleotidetriphosphates. This can be done for example by increasing the viscosityin the reaction chamber. This can be accomplished by adding to thereaction chamber a viscosity increasing agent such as but not limited topolyethylene glycol (PEG) or PEA. Diffusion can also be delayed by theuse of packing beads in the reaction wells. In some embodiments of theinvention, the time for a 4 nucleotide cycle may be 50-100 seconds,60-90 seconds, or about 70 seconds. In other embodiments, this cycletime can be equal to or less than 70 seconds, including equal to or lessthan 60 seconds, equal to or less than 50 seconds, equal to or less than40 seconds, or equal to or less than 30 seconds. A read length of about400 bases may take on the order of 30 minutes, 60 minutes, 1.5 hours, 2hours, 2.5 hours, 3 hours, 3.5 hours, 4 hours, 4.5 hours, or in someinstance 5 or more hours. These times are sufficient for the sequencingof megabases, and more preferably gigabases of sequence, with greateramounts of sequence being attainable through the use of denser arrays(i.e., arrays with greater numbers of reaction wells and FETs) and/orthe simultaneous use of multiple arrays.

Table 2 provides estimates for the rates sequencing based on variousarray, chip and system configurations contemplated herein. It is to beunderstood that the invention contemplates even denser arrays than thoseshown in Table 2. These denser arrays can be characterized as 90 nm CMOSwith a pitch of 1.4 μm and a well size of 1 μm which may be used with0.7 μm beads, or 65 nm CMOS with a pitch of 1 μm and a well size of 0.5μm which may be used with 0.3 μm beads, or 45 μm CMOS with a pitch of0.7 μm and a well size of 0.3 μm which can be used with 0.2 μm beads.

TABLE 2 Reaction Parameters and Read Rates. chip type A B C D Epixel/CMOS 2.8 um/0.18 um 5.1 um/0.35 um 5.1 um/0.35 um 9 um/3.5 um 9um/0.35 um chip size 17.5 × 17.5 17.5 × 17.5 12 × 12 17.5 × 17.5 12 × 12# possible reads 27,800,000 7,220,000 2,950,000 2,320,000 1,060,000 readlength (assumption) 400 400 400 400 400 # chips/board 4 4 4 4 4 beadload efficiency 0.80 0.80 0.80 0.80 0.80 # yielded Gbp* per run 35.6 9.23.8 3.0 1.4 # times HG** (3 Gbp/HG) 11.86 3.08 1.26 0.99 0.45 *Gbp isgigabases **HG is human genome

The template nucleic acid is also contacted with other reagents and/orcofactors including but not limited to buffer, detergent, reducingagents such as dithiothrietol (DTT, Cleland's reagent), single strandedbinding proteins, and the like before and/or while in the well. In oneembodiment, the polymerase comprises one or more single stranded bindingproteins (e.g., the polymerase may be one that is engineered to includeone or more single stranded binding proteins). In one embodiment, thetemplate nucleic acid is contacted with the primer and the polymeraseprior to its introduction into the flow chamber and wells thereof.

In one embodiment, the nucleic acid loaded beads are introduced into theflow chamber and ultimately the wells situated above the chemFET array.The method requires that each well in the flow chamber contain only onenucleic acid loaded bead since the presence of two beads per well willyield unusable sequencing information derived from two differenttemplate nucleic acids. The Examples provides a brief description of anexemplary bead loading protocol in the context of magnetic beads. It isto be understood that a similar approach could be used to load otherbead types. The protocol has been demonstrated to reduce the likelihoodand incidence of trapped air in the wells of the flow chamber, uniformlydistribute nucleic acid loaded beads in the totality of wells of theflow chamber, and avoid the presence and/or accumulation of excess beadsin the flow chamber. In some embodiments, the microwell array may beanalyzed to determine the degree of loading of beads into themicrowells, and in some instances to identify those microwells havingbeads and those lacking beads. The ability to know which microwells lackbeads provides another internal control for the sequencing reaction. Thepresence or absence of a bead in a well can be determined by standardmicroscopy or by the sensor itself. FIGS. 61A and B are images capturedfrom an optical microscope inspection of a microwell array (A) and fromthe sensor array underlying the microwell array (B). The white spots inboth images each represent a bead in a well. Such microwell observationusually is only made once per run particularly since the beads oncedisposed in a microwell are unlikely to move to another well.

The percentage of occupied wells in the well array may vary depending onthe methods being performed. If the method is aimed at extractingmaximum sequence data in the shortest time possible, then higheroccupancy is desirable. If speed and throughout is not as critical, thenlower occupancy may be tolerated. Therefore depending on the embodiment,suitable occupancy percentages may be at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99%, or 100% of the wells. As used herein, occupancy refers to thepresence of one nucleic acid loaded bead in a well and the percentageoccupancy refers to the proportion of total wells in an array that areoccupied by a single bead. Wells that are occupied by more than one beadcannot be used in the analyses contemplated by the invention.

Ultimately a homogeneous population of template nucleic acids is placedinto each of a plurality of wells, each well situated over and thuscorresponding to at least one sensor. As discussed above, preferably thewell contains at least 10, at least 100, at least 1000, at least 10⁴, atleast 10⁵, at least 10⁶, or more copies of an identical template nucleicacid. Identical template nucleic acids means that the templates areidentical in sequence. Most and preferably all the template nucleicacids within a well are uniformly hybridized to a primer. Uniformhybridization of the template nucleic acids to the primers means thatthe primer hybridizes to the template at the same location (i.e., thesequence along the template that is complementary to the primer) asevery other template/primer hybrid in the well. The uniform positioningof the primer on every template allows the coordinated synthesis of allnew nucleic acid strands within a well, thereby resulting in a greatersignal-to-noise ratio.

In some embodiments, nucleotides are then added in flow, or by any othersuitable method, in sequential order to the flow chamber and thus thewells. The nucleotides can be added in any order provided it is knownand for the sake of simplicity kept constant throughout a run.

As mentioned herein, in some embodiments, the method relies on detectionof PPi and unincorporated nucleotides. In other embodiments,unincorporated nucleotides are not detected. This can be accomplishedfor example by adding ATP to the wash buffer so that dNTPs flowing intoa well displace ATP from the well. The ATP matches the ionic strength ofthe dNTPs entering the wells and it also has a similar diffusion profileas those dNTPs. In this way, influx and efflux of dNTPs during thesequencing reaction do not interfere with measurements at the chemFET.The concentration of ATP used is on the order of the concentration ofdNTP used.

In some embodiments, particularly those in which a low ionic strengthenvironment is used, the dNTP and/or the polymerase will bepre-incubated with divalent cation such as but not limited to Mg²⁺ (forexample in the form of MgCl₂) or Mn²⁺ (for example in the form ofMnCl₂). Other divalent cations can also be used including but notlimited to Ca²⁺, Co²⁺. This pre-incubation (and thus “pre-loading” ofthe dNTP and/or the polymerase can ensure that the polymerase is exposedto a sufficient amount of divalent cation for proper and necessaryfunctioning even if it is present in a low ionic strength environment.Pre-incubation may occur for 1-60 minutes, 5-45 minutes, or 10-30minutes, depending on the embodiment, although the invention is notlimited to these time ranges.

A sequencing cycle may therefore proceed as follows washing of the flowchamber (and wells) with wash buffer (optionally containing ATP),introduction of a first dNTP species (e.g., dATP) into the flow chamber(and wells), release and detection of PPi and then unincorporatednucleotides (if incorporation occurred) or detection of solelyunincorporated nucleotides (if incorporation did not occur) (by any ofthe mechanisms described herein), washing of the flow chamber (andwells) with wash buffer, washing of the flow chamber (and wells) withwash buffer containing apyrase (to remove as many of the unincorporatednucleotides as possible prior to the flow through of the next dNTP,washing of the flow chamber (and wells) with wash buffer, andintroduction of a second dNTP species. This process is continued untilall 4 dNTP (i.e., dATP, dCTP, dGTP and dTTP) have been flowed throughthe chamber and allowed to incorporate into the newly synthesizedstrands. This 4-nucleotide cycle may be repeated any number of timesincluding but not limited to 10, 25, 50, 100, 200 or more times. Thenumber of cycles will be governed by the length of the template beingsequenced and the need to replenish reaction reagents, in particular thedNTP stocks and wash buffers.

As part of the sequencing reaction, a dNTP will be ligated to (or“incorporated into” as used herein) the 3′ of the newly synthesizedstrand (or the 3′ end of the sequencing primer in the case of the firstincorporated dNTP) if its complementary nucleotide is present at thatsame location on the template nucleic acid. Incorporation of theintroduced dNTP (and concomitant release of PPi) therefore indicates theidentity of the corresponding nucleotide in the template nucleic acid.If no dNTP has been incorporated, then only a single ion pulse isdetected corresponding to unincorporated nucleotides at the surface ofthe chemFET. One can therefore conclude that the complementarynucleotide was not present in the template at that location. If theintroduced dNTP has been incorporated into the newly synthesized strand,then the chemFET will detect a first ion pulse corresponding to the PPiand a second pulse corresponding to the unincorporated nucleotides. Itis further possible to quantitate the number of dNTP incorporated by forexample measuring the time difference between the time for of the first(PPi) ion pulse (i.e., t₀) and the second (unincorporated nucleotides)ion pulse (i.e., t₁). The greater the time difference, the greater thenumber of nucleotides incorporated. The result is that no sequenceinformation is lost through the sequencing of a homopolymer stretch(e.g., poly A, poly T, poly C, or poly G) in the template.

Apyrase is an enzyme that degrades residual unincorporated nucleotidesconverting them into monophosphate and releasing inorganic phosphate inthe process. It is useful for degrading dNTPs that are not incorporatedand/or that are in excess. It is important that excess and/orunincorporated dNTP be washed away from all wells after measurements arecomplete and before introduction of the subsequent dNTP. Accordingly,addition of apyrase between the introduction of different dNTPs isuseful to remove unincorporated dNTPs that would otherwise obscure thesequencing data.

Additional sequencing reaction reagents such as those described abovemay be introduced throughout the reaction, although in some cases thismay not be necessary. For example additional polymerase, DTT, SBB andthe like may be added if necessary.

The invention therefore contemplates performing a plurality of differentsequencing reactions simultaneously. A plurality of identical sequencingreactions is occurring in each occupied well simultaneously. It is thissimultaneous and identical incorporation of dNTP within each well thatincreases the signal to noise ratio. By performing sequencing reactionsin a plurality of wells simultaneously, a plurality of different nucleicacids are simultaneously sequenced.

The methods aim to maximize complete incorporation across all microwellsfor any given dNTP, reduce or decrease the number of unincorporateddNTPs that remain in the wells after signal detection is complete, andachieve as a high a signal to noise ratio as possible.

The sequencing reaction can be run at a range of temperatures.Typically, the reaction is run in the range of 30-60° C., 35-55° C., or40-45° C. It is preferable to run the reaction at temperatures thatprevent formation of secondary structure in the nucleic acid. Howeverthis must be balanced with the binding of the primer (and the newlysynthesized strand) to the template nucleic acid and the reducedhalf-life of apyrase at higher temperatures. A suitable temperature isabout 41° C. The solutions including the wash buffers and the dNTPsolutions are generally warmed to these temperatures in order not toalter the temperature in the wells. The wash buffer containing apyrasehowever is preferably maintained at a lower temperature in order toextend the half-life of the enzyme. Typically, this solution ismaintained at about 4-15° C., and more preferably 4-10° C. It will beappreciated that sequencing in a low ionic concentration can lower theTm of the sequencing primer, possibly below that optimal for thepolymerase. The invention therefore contemplates that sequencing primersmay comprise nucleotide modifications such as modified bases, linkednucleic acid (LNA) monomers, peptide nucleic acid (PNA) monomers, oralternatively minor groove binders could be used to elevate the primerTm into a suitable range. The nucleotide incorporation reaction canoccur very rapidly. As a result, it may be desirable in some instancesto slow the reaction down or to slow the diffusion of analytes in thewell in order to ensure maximal data capture during the reaction. Thediffusion of reagents and/or byproducts can be slowed down in a numberof ways including but not limited to addition of packing beads in thewells, and/or the use of polymers such as polyethylene glycol in thewells (e.g., PEG attached to the capture beads and/or to packing beads).The packing beads also tend to increase the concentration of reagentsand/or byproducts at the chemFET surface, thereby increasing thepotential for signal. The presence of packing beads generally allows agreater time to sample (e.g., by 2- or 4-fold).

Data capture rates can vary and be for example anywhere from 10-100frames per second and the choice of which rate to use will be dictatedat least in part by the well size and the presence of packing beads orother diffusion limiting techniques. Smaller well sizes generallyrequire faster data capture rates.

In some aspects of the invention that are flow-based and where the topface of the well is open and in communication with fluid over theentirety of the chip, it is important to detect the released PPi orother byproduct (e.g., H⁺) prior to its diffusion out of the well.Diffusion of reaction byproducts out of the well will lead to falsenegatives (because the byproduct is not detected in that well) andpotential false positives in adjacent or downstream wells (where thebyproduct may be detected), and thus should be avoided. Packing beadsand/or polymers such as PEG may also help reduce the degree of diffusionand/or cross-talk between wells.

Thus in some embodiments packing beads are used in addition to thenucleic acid-loaded beads. The packing beads may be magnetic (includingsuperparamagnetic) but they are not so limited. In some embodiments thepacking beads and the capture beads are made of the same material (e.g.,both are magnetic, both are polystyrene, etc.), while in otherembodiment they are made of different materials (e.g., the packing beadsare polystyrene and the capture beads are magnetic). The packing beadsare generally smaller than the capture beads. The difference in size maybe vary and may be 5-fold, 10-fold, 15-fold, 20-fold or more. As anexample, 0.35 μm diameter packing beads can be used with 5.91 μm capturebeads. Such packing beads are commercially available from sources suchas Bang Labs. The placement of the packing beads relative to the capturebead may vary. For example, the packing beads may surround the capturebead and thereby prevent the capture bead from contacting the chemFETsurface. As another example, the packing beads may be loaded into thewells following the capture beads in which case the capture bead is incontact with the chemFET surface. The presence of packing beads betweenthe capture bead and the chemFET surface slows the diffusion of thesequencing byproducts such as PPi, thereby facilitating data capture.

The invention further contemplates the use of packing beads ormodifications to the chemFET surface (as described herein) to preventcontact and thus interference of the chemFET surface with the templatenucleic acids bound to the capture beads. A layer of packing beads thatis 0.1-0.5 μm in depth or height would preclude this interaction.

The sequencing reaction may be preceded by an analysis of the arrays todetermine the location of beads. It has been found that in the absenceof flow the background signal (i.e., noise) is less than or equal toabout 0.25 mV, but that in the presence of DNA-loaded capture beads thatsignal increases to about 1.0 mV=/−0.5 mV. This increase is sufficientto allow one to determine wells with beads.

The invention further contemplates kits comprising the various reagentsnecessary to perform a sequencing reaction and instructions of useaccording to the methods set forth herein.

One preferred kit comprises one or more containers housing wash buffer,one or more containers each containing one of the following reagents:dATP buffer, dCTP buffer, dGTP buffer or dTTP buffer, dATP, dCTP, dGTPand dTTP stocks, apyrase, SSB, polymerase, packing beads, and optionallypyrophosphatase. Importantly the kits may comprise only naturallyoccurring dNTPs. The kits may also comprise one or more wash bufferscomprising components as described in the Examples, but are not solimited. The kits may also comprise instructions for use includingdiagrams that demonstrate the methods of the invention.

It is to be understood that interactions between receptors and ligandsor between two members of a binding pair or between components of amolecular complex can also be detected using the chemFET arrays.Examples of such interactions include hybridization of nucleic acids toeach other, protein-nucleic acid binding, protein-protein binding,enzyme-substrate binding, enzyme-inhibitor binding, antigen-antibodybinding, and the like. Any binding or hybridization event that causes achange of the semiconductor charge density at the FET interface and thuschanges the current that flows from the source to the drain of thesensors described herein can be detected according to the invention.

In these embodiments, the passivation layer (or possibly an intermediatelayer coated onto the passivation layer) is functionalized with nucleicacids (e.g., DNA, RNA, miRNA, cDNA, and the like), antigens (which canbe of any nature), proteins (e.g., enzymes, cofactors, antibodies,antibody fragments, and the like), and the like. Conjugation of theseentities to the passivation layer can be direct or indirect (e.g., usingbifunctional linkers that bind to both the passivation layer reactivegroup and the entity to be bound).

As an example, reaction groups such as amine or thiol groups may beadded to a nucleic acid at any nucleotide during synthesis to provide apoint of attachment for a bifunctional linker. As another example, thenucleic acid may be synthesized by incorporating conjugation-competentreagents such as Uni-Link AminoModifier, 3′-DMT-C6-Amine-ON CPG,AminoModifier II, N-TFA-C6-AminoModifier, C6-ThiolModifier, C6-DisulfidePhosphoramidite and C6-Disulfide CPG (Clontech, Palo Alto, Calif.).Other methods for attaching nucleic acids are discussed below.

In one aspect of the invention, the chemFET arrays are provided incombination with nucleic acid arrays. Nucleic acids in the form of shortnucleic acids (e.g., oligonucleotides) or longer nucleic acids (e.g.,full length cDNAs) can be provided on chemFET surfaces of the arraysdescribed herein. Nucleic acid arrays generally comprise a plurality ofphysically defined regions on a planar surface (e.g., “spots”) each ofwhich has conjugated to it one and more preferably more nucleic acids.The nucleic acids are usually single stranded. The nucleic acidsconjugated to a given spot are usually identical. In the context of anoligonucleotide array, these nucleic acids may be on the order of less100 nucleotides in length (including about 10, 20, 25, 30, 40, 50, 60,70, 80, 90 or 100 nucleotides in length). If the arrays are used todetect certain genes (including mutations in such genes or expressionlevels of such genes), then the array may include a number of spots eachof which contains oligonucleotides that span a defined and potentiallydifferent sequence of the gene. These spots are then located across theplanar surface in order to exclude position related effects in thehybridization and readout means of the array.

The arrays are contacted with a sample being tested. The sample may be agenomic DNA sample, a cDNA sample from a cell, a tissue or a mass (e.g.,a tumor), a population of cells that are grown on the array, potentiallyin a two dimensional array that corresponds to the underlying sensorarray, and the like. Such arrays are therefore useful for determiningpresence and/or level of a particular gene or of its expression,detecting mutations within particular genes (such as but not limited todeletions, additions, substitutions, including single nucleotidepolymorphisms), and the like.

The binding or hybridization of the sample nucleic acids and theimmobilized nucleic acids is generally performed under stringenthybridization conditions as that term is understood in the art. (See forexample Sambrook et al. “Maniatis”.) Examples of relevant conditionsinclude (in order of increasing stringency): incubation temperatures of25° C., 37° C., 50° C. and 68° C.; buffer concentrations of 10×SSC,6×SSC, 4×SSC, 1×SSC, 0.1×SSC (where SSC is 0.15 M NaCl and 15 mM citratebuffer) and their equivalents using other buffer systems; formamideconcentrations of 0%, 25%, 50%, and 75%; incubation times from 5 minutesto 24 hours; 1, 2, or more washing steps; wash incubation times of 1, 2,or 15 minutes; and wash solutions of 6×SSC, 1×SSC, 0.1×SSC, or deionizedwater. By way of example hybridization may be performed at 50% formamideand 4×SSC followed by washes of 2×SSC/formamide at 50° C. and with1×SSC.

Nucleic acid arrays include those in which already formed nucleic acidssuch as cDNAs are deposited (or “spotted”) on the array in a specificlocation. Nucleic acids can be spotted onto a surface bypiezoelectrically deposition, UV crosslinking of nucleic acids topolymer layers such as but not limited to poly-L-lysine or polypyrrole,direct conjugation to silicon coated SiO₂ as described in published USpatent application 2003/0186262, direct conjugation to a silanisedchemFET surface (e.g., a surface treated with3-aminopropyltriethoxysilane (APTES) as described by Uslu et al.Biosensors and Bioelectronics 2004, 19:1723-1731, for example.

Nucleic acid arrays also include those in which nucleic acids (such asoligonucleotides of known sequence) are synthesized directly on thearray. Nucleic acids can be synthesized on arrays using art-recognizedtechniques such as but not limited to printing with fine-pointed pinsonto glass slides, photolithography using pre-made masks,photolithography using dynamic micromirror devices (such as DLPmirrors), ink-jet printing, or electrochemistry on microelectrodearrays. Reference can also be made to Nuwaysir et al. 2002 “Geneexpression analysis using oligonucleotide arrays produced by masklessphotolithography.”. Genome Res 12: 1749-1755. Commercial sources of thislatter type of array include Agilent, Affymetrix, and NimbleGen. Thusthe chemFET passivation layer may be coated with an intermediate layerof reactive molecules (and therefore reactive groups) to which thenucleic acids are bound and/or from which they are synthesized.

The invention contemplates combining such nucleic acid arrays with thechemFET arrays and particularly the “large scale” chemFET arraysdescribed herein. The chemFET/nucleic acid array can be used in avariety of applications, some of which will not require the wells (ormicrowells or reaction chambers, as they are interchangeably referred toherein). Since analyses may still be carried out in flow, including in a“closed” system (i.e., where the flow of reagents and wash solutions andthe like is automated), there will be one or more flow chambers situatedabove and in contact with the array. The use of multiple flow chambersallows multiple samples (or nucleic acid libraries) to be analyzedsimultaneously. There may be 2, 3, 4, 5, 6, 7, 8, 9, 10 or more flowchambers. This configuration applies equally to other biological arraysincluding those discussed herein such as protein arrays, antibodyarrays, enzyme arrays, chemical arrays, and the like.

Since the binding event between binding partners or between componentsof a complex is detected electronically via the underlying FET, suchassays may be carried out without the need to manipulate (e.g.,extrinsically label) the sample being assayed. This is advantageoussince such manipulation invariably results in loss of sample andgenerally requires increased time and work up. In addition, the presentmethod allows binding interactions to be studied in real time.

Protein arrays used in combination with the chemFET arrays of theinvention are also contemplated. Protein arrays comprise proteins orpeptides or other amino acid comprising biological moiety bound to aplanar surface in an organized and predetermined manner. Such proteinsinclude but are not limited to enzymes, antibodies and antibodyfragments or antibody mimics (e.g., single chain antibodies).

In one embodiment, a protein array may comprise a plurality of differentproteins (or other amino acid containing biological moieties). Eachprotein, and preferably a plurality of proteins, is present in apredetermined region or “cell” of the array. The regions (or cells) arealigned with the sensors in the sensor array such that there is onesensor for each region (or cell). The plurality of proteins in a singleregion (or cell) may vary depending on the size of the protein and thesize of the region (or cell) and may be but is not limited to at least10, 50, 100, 500, 10³, 10⁴ or more. The array itself may have any numberof cells, including but not limited to at least 10, 10², 10³, 10⁴, 10⁵,10⁶, 10⁷, or more. In one application, the array is exposed to a samplethat is known to contain or is suspected of containing an analyte thatbinds to the protein. The analyte may be a substrate or an inhibitor ifthe protein is an enzyme. The analyte may be any molecule that binds tothe protein including another protein, a nucleic acid, a chemicalspecies (whether synthetic or naturally occurring), and the like.

It is to be understood that, like the nucleic acid arrays contemplatedherein, the readout from the protein arrays will be a change in currentthrough the chemFET and thus no additional step of labeling and/or labeldetection is required in these array methods.

In another embodiment, the protein array may comprise a plurality ofidentical proteins (or other amino acid containing biological moieties).The identical proteins may be uniformly distributed on a planar surfaceor they may be organized into discrete regions (or cells) on thatsurface. In these latter embodiments, the regions (or cells) are alignedwith the sensors in the sensor array such that there is one sensor foreach region (or cell).

The proteins may be synthesised off-chip, then purified and attached tothe array. Alternatively they can be synthesised on-chip, similarly tothe nucleic acids discussed above. Synthesis of proteins using cell-freeDNA expression or chemical synthesis is amenable to on-chip synthesis.Using cell-free DNA expression, proteins are attached to the solidsupport once synthesized. Alternatively, proteins may be chemicallysynthesized on the solid support using solid phase peptide synthesis.Selective deprotection is carried out through lithographic methods or bySPOT-synthesis. Reference can be made to at least MacBeath andSchreiber, Science, 2000, 289:1760-1763, or Jones et al. Nature, 2006,439:168-174. Reference can also be made to U.S. Pat. No. 6,919,211 toFodor et al.

Chemical compound microarrays in combination with chemFET arrays arealso envisioned. Chemical compound microarrays can be made by covalentlyimmobilizing the compounds (e.g., organic compounds) on the solidsurface with diverse linking techniques (may be referred to in theliterature as “small molecule microarray”), by spotting and dryingcompounds (e.g., organic compounds) on the solid surface withoutimmobilization (may be referred to in the literature as “micro arrayedcompound screening (μARCS)”), or by spotting organic compounds in ahomogenous solution without immobilization and drying effect(commercialized as DiscoveryDot™ technology by Reaction BiologyCorporation).

Tissue microarrays in combination with chemFET arrays are furthercontemplated by the invention. Tissue microarrays are discussed ingreater detail in Battifora Lab Invest 1986, 55:244-248; Battifora andMehta Lab Invest 1990, 63:722-724; and Kononen et al. Nat Med 1998,4:844-847.

The configurations of the chemFET arrays and the biological or chemicalarrays are similar in each instance and the discussion of onecombination array will apply to others described herein or otherwiseknown in the art.

In yet another aspect, the invention contemplates analysis of cellcultures (e.g., two-dimensional cells cultures) (see for example Baumannet al. Sensors and Actuators B 55 1999 77:89), and tissue sectionsplaced in contact with the chemFET array. As an example, a brain sectionmay be placed in contact with the chemFET array of the invention andchanges in the section may be detected either in the presence or absenceof stimulation such as but not limited to neurotoxins and the like.Transduction of neural processes and/or stimulation can thereby beanalyzed. In these embodiments, the chemFETs may operate by detectingcalcium and/or potassium fluxes via the passivation layer itself or viareceptors for these ions that are coated onto the passivation layer.

In yet another aspect, the invention contemplates the use of chemFETarrays, functionalized as described herein or in another manner, for usein vivo. Such an array may be introduced into a subject (e.g., in thebrain or other region that is subject to ion flux) and then analyzed forchanges based on the status of the subject.

Methods for attaching nucleic acids, proteins, molecules, and the liketo solid supports, particularly in the context of an array, have beendescribed in the art. See for example Lipshutz et al. Nat. Genet.(supplement) 1999 21:20-24; Li et al. Proc. Natl. Acad. Sci., 2001,98:31-36; Lockhart et al. Nat. Biotechnol. 1996 14:1675-1680; Wodicka etal. Nat. Biotechnol. 1997 15: 1359-1367; Chen et al. Journal ofBiomedical Optics 1997 2:364; Duggan et al. Nat Genet. 1991 21(1Suppl):10-4; Marton et al. Nat. Med. 1998 4(11):1293-301; Kononen et al.Nat Med 1998 4(7):844-847; MacBeath et al., Science 2000289(5485):1760-1763; Haab et al. Genome Biology 2001 2(2); Pollack etal. Nat Genet. 1999 23(1):41-6; Wang D G et al. Science 1998280(5366):1077-82; Fodor et al. Science 1991 251:767-773; Fodor et al.Nature 1993 364:555-556; Pease et al. Proc. Natl. Acad. Sci. USA 199491:5022-5026; Fodor Science 1997 277:393-395; Southern et al. Genomics1992 13: 1008-1017; Schena et al. Science 1995 270(5235):467-70; Shalonet al. Genome Res 1996 6(7):639-45; Jongsma Proteomics 2006,6:2650-2655; Sakata, Biosensors and Bioelectronics 2007, 22: 1311-1316.

The systems described herein can be used for sequencing unlabeledbiological polymers without optical detection.

In some embodiments, the invention encompasses a sequencing apparatusadapted for sequencing unlabeled biological polymers without opticaldetection and comprising an array of at least 100 reaction chambers.

Typically, each reaction chamber is capacitively coupled to a chemFET.

Preferably, each reaction chamber is no greater than about 0.39 pL involume and about 49 μm² surface aperture, and more preferably has anaperture no greater than about 16 μm² and volume no greater than about0.064 pL. Preferably, the array has at least 1,000, 10,000, 100,000, or1,000,000 reaction chambers.

Typically, the reaction chambers comprise microfluidic wells.

Preferably, the apparatus is adapted to sequence at least 10⁶ base pairsper hour, more preferably at least 10⁷ base pairs per hour, and mostpreferably at least 10⁸ base pairs per hour.

In another embodiment, the invention encompasses a method for sequencinga biological polymer with the above-described apparatus comprisingmeasuring time of incorporation of individual monomers into anelongating polymer.

Typically, the biological polymer is a nucleic acid template and themonomer is a nucleotide. Preferably, the nucleic acid template has200-700 base pairs. Preferably, the nucleic acid template is amplifiedprior to determining the sequence.

Typically, the measuring is performed under diffusion limitedconditions. The measuring may comprise detecting an electrical change,detecting an ion pulse, or detecting the release of inorganicpyrophosphate (“PPi”). Preferably, the incorporation takes place at anionic strength no greater than 400 μM, wherein the concentration of Mg²⁺or Mn²⁺ or other divalent cation is no greater than 100 μM. Preferably,at least 10⁶ base pairs are sequenced per hour, more preferably at least10⁷ base pairs are sequenced per hour, and most preferably at least 10⁸base pairs are sequenced per hour using the above-described method.Preferably the detecting is accomplished by an array of chemFETs asabove described.

In some embodiments, the invention encompasses a method for determininga nucleic acid sequence comprising: performing at least 100 sequencingreactions simultaneously; and determining the sequence without the useof labels and without the use of optical detection.

The nucleic acid template used in this and other methods of theinvention may be derived from a variety of sources by a variety ofmethods, all known to those of ordinary skill in the art. Templates maybe derived from, but are not limited to, entire genomes of varyingcomplexity, cDNA, mRNA or siRNA samples, or may represent entirepopulations, as in the various environmental and metabiome sequencingprojects. Template nucleic acids may also be generated from specificsubsets of nucleic acid populations including but not limited to PCRproducts, specific exons or regions of interest, or 16S or otherdiagnostic or identifying genomic regions.

Typically, the required starting material for sequencing is less than 3μg of nucleic acids. Preferably, the template nucleic acid is amplifiedprior to sequencing. The template nucleic acid may be optionally boundto one or more beads prior to sequencing.

Typically, the determining step comprises measuring the amount of timeit takes for one or more of a first monomer to incorporate into anelongating sequence. Typically, the incorporation of the monomer ismeasured under diffusion limited conditions. The measuring may comprisedetecting an electrical change, detecting an ion pulse, or detecting therelease of inorganic pyrophosphate (“PPi”).

Preferably, at least 10⁶ base pairs are sequenced per hour, morepreferably at least 10⁷ base pairs are sequenced per hour, and mostpreferably at least 10⁸ base pairs are sequenced per hour using theabove-described method. Thus, the method may be used to sequence anentire human genome within about 24 hours, more preferably within about20 hours, even more preferably within about 15 hours, even morepreferably within about 10 hours, even more preferably within about 5hours, and most preferably within about 1 hour. These rates may beachieved using multiple ISFET arrays as shown herein, and processingtheir outputs in parallel.

The following examples are included for purposes of illustration and arenot intended to limit the scope of the invention.

EXAMPLES Example 1 Bead Preparation

Binding of Single-Stranded Oligonucleotides to Streptavidin-CoatedMagnetic Beads. Single-stranded DNA oligonucleotide templates with a 5′Dual Biotin tag (HPLC purified), and a 20-base universal primer wereordered from IDT (Integrated DNA Technologies, Coralville, Ind.).Templates were 60 bases in length, and were designed to include 20 basesat the 3′ end that were complementary to the 20-base primer (Table 3,italics). The lyophilized and biotinylated templates and primer werere-suspended in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8) as 40 μMstock solutions and as a 400 μM stock solution, respectively, and storedat −20° C. until use.

For each template, 60 μl of magnetic 5.91 μm (Bangs Laboratories, Inc.Fishers, Ind.) streptavidin-coated beads, stored as an aqueous, bufferedsuspension (8.57×10⁴ beads/μL), at 4° C., were prepared by washing with120 μl bead wash buffer three times and then incubating with templates1, 2, 3 and 4 (T1, T2, T3, T4: Table 3) with biotin on the 5′ end,respectively.

Due to the strong covalent binding affinity of streptavidin for biotin(Kd˜10-15), these magnetic beads are used to immobilize the templates ona solid support, as described below. The reported binding capacity ofthese beads for free biotin is 0.650 pmol/μL of bead stock solution. Fora small (<100 bases) biotinylated ssDNA template, it was conservativelycalculated that 9.1×10⁵ templates could be bound per bead. The beads areeasily concentrated using simple magnets, as with the Dynal MagneticParticle Concentrator or MPC-s (Invitrogen, Carlsbad, Calif.). The MPC-swas used in the described experiments.

An MPC-s was used to concentrate the beads for 1 minute between eachwash, buffer was then added and the beads were resuspended. Followingthe third wash the beads were resuspended in 120 μL bead wash bufferplus 1 μl of each template (40 μM). Beads were incubated for 30 minuteswith rotation (Labquake Tube Rotator, Barnstead, Dubuque, Iowa).Following the incubation, beads were then washed three times in 120 μLAnnealing Buffer (20 mM Tris-HCl, 5 mM magnesium acetate, pH 7.5), andre-suspended in 60 μL of the same buffer.

TABLE 3 Sequences for Templates 1, 2, 3, and 4T1: 5′/52Bio/GCA AGT GCC CTT AGG CTT CAG TTC AAA AGT CCT AAC TGG GCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 1)T2: 5′/52Bio/CCA TGT CCC CTT AAG CCC CCC CCA TTC CCC CCT GAA CCC CCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 2)T3: 5′/52Bio/AAG CTC AAA AAC GGT AAA AAA AAG CCA AAA AAC TGG AAA ACA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 3)T4: 5′/52Bio/TTC GAG TTT TTG CCA TTT TTT TTC GGT TTT TTG ACC TTT TCA AGG CAC ACA GGG GAT AGG-3′ (SEQ ID NO: 4)

Annealing of Sequencing Primer. The immobilized templates, bound at the5′ end to 5.91 μm magnetic beads, are then annealed to a 20-base primercomplementary to the 3′ end of the templates (Table 3). A 1.0 μL aliquotof the 400 μM primer stock solution, representing a 20-fold excess ofprimer to immobilized template, is then added and then the beads plustemplate are incubated with primer for 15 minutes at 95° C. and thetemperature was then slowly lowered to room temperature. The beads werethen washed 3 times in 120 of 25 mM Tricine buffer (25 mM Tricine, 0.4mg/ml PVP, 0.1% Tween 20, 8.8 mM Magnesium Acetate; ph 7.8) as describedabove using the MPC-s. Beads were resuspended in 25 mM Tricine buffer.

Incubation of Hybridized Templates/Primer with DNA Polymerase. Templateand primer hybrids are incubated with polymerase essentially asdescribed by Margulies et al. Nature 2005 437(15):376-380 andaccompanying supplemental materials.

Loading of Prepared Test Samples onto the ISFET Sensor Array. Thedimensions and density of the ISFET array and the microfluidicspositioned thereon may vary depending on the application. A non-limitingexample is a 512×512 array. Each grid of such an array (of which therewould be 262144) has a single ISFET. Each grid also has a well (or asthey may be interchangeably referred to herein as a “microwell”)positioned above it. The well (or microwell) may have any shapeincluding columnar, conical, square, rectangular, and the like. In oneexemplary conformation, the wells are square wells having dimensions of7×7×10 μm. The center-to-center distance between wells is referred toherein as the “pitch”. The pitch may be any distance although it ispreferably to have shorter pitches in order to accommodate as large ofan array as possible. The pitch may be less than 50 μm, less than 40 μm,less than 30 μm, less than 20 μm, or less than 10 μm. In one embodiment,the pitch is about 9 μm. The entire chamber above the array (withinwhich the wells are situated) may have a volume of equal to or less thanabout 30 μL, equal to or less than about 20 μL, equal to or less thanabout 15 μL, or equal to or less than 10 μL. These volumes thereforecorrespond to the volume of solution within the chamber as well.

Loading of Beads in an ‘Open’ System. Beads with templates 1-4 wereloaded on the chip (10 μL of each template). Briefly, an aliquot of eachtemplate was added onto the chip using an Eppendorf pipette. A magnetwas then used to pull the beads into the wells.

Loading of Beads in a ‘Closed’ System. Both the capture beads thepacking beads are loaded using flow. Microliter precision of beadsolution volume, as well as positioning of the bead solution through thefluidics connections, is achieved as shown in FIGS. 62-70 using the beadloading fitting, which includes a major reservoir (approx. 1 mL involume), minor reservoir (approx. 10 μL in volume), and a microfluidicchannel for handling small volumes of bead solution. This method alsoleverages the microliter precision of fluid application allowed byprecision pipettes.

The chip comprising the ISFET array and flow cell is seated in the ZIF(zero insertion force) socket of the loading fixture, then attaching astainless steel capillary to one port of the flow cell and flexiblenylon tubing on the other port. Both materials are microfluidic-typefluid paths (e.g., on the order of <0.01″ inner diameter). The beadloading fitting, consisting of the major and minor reservoirs, itattached to the end of the capillary. A common plastic syringe is filledwith buffer solution, then connected to the free end of the nylontubing. The electrical leads protruding from the bottom of the chip areinserted into a socket on the top of a fixture unit (not shown).

The chip comprising the ISFET array and flow cell is seated in a socketsuch as a ZIF (zero insertion force) socket of the loading fixture, thena stainless steel capillary may be attached to one port of the flow celland flexible nylon tubing on the other port. Both materials aremicrofluidic-type fluid paths (e.g., on the order of <0.01″ innerdiameter). The bead loading fitting, consisting of the major and minorreservoirs, it attached to the end of the capillary. A common plasticsyringe is filled with buffer solution, then connected to the free endof the nylon tubing. The electrical leads protruding from the bottom ofthe chip are inserted into a socket on the top of a fixture unit (notshown).

It will be appreciated that there will be other ways of drawing thebeads into the wells of the flow chamber, including centrifugation orgravity. The invention is not limited in this respect.

DNA Sequencing using the ISFET Sensor Array in an Open System. Aillustrative sequencing reaction can be performed in an ‘open’ system(i.e., the ISFET chip is placed on the platform of the ISFET apparatusand then each nucleotide (5 μL resulting in 6.5 μM each) is manuallyadded in the following order: dATP, dCTP, dGTP and dTT (100 mM stocksolutions, Pierce, Milwaukee, Wis.), by pipetting the given nucleotideinto the liquid already on the surface of the chip and collecting datafrom the chip at a rate of 2.5 mHz. This can result in data collectionover 7.5 seconds at approximately 18 frames/second. Data may thenanalyzed using LabView.

Given the sequences of the templates, it is expected that addition ofdATP will result in a 4 base extension for template 4. Addition of dCTPwill result in a 4 base extension in template 1. Addition of dGTP willcause template 1, 2 and 4 to extend as indicated in Table 4 and additionof dTTP will result in a run-off (extension of all templates asindicated).

Preferably when the method is performed in a non-automated manner (i.e.,in the absence of automated flow and reagent introduction), each wellcontains apyrase in order to degrade the unincorporated dNTPs, oralternatively apyrase is added into each well following the addition andincorporation of each dNTP (e.g., dATP) and prior to the addition ofanother dNTP (e.g., dTTP). It is to be understood that apyrase can besubstituted, in this embodiment or in any other embodiment discussedherein, with another compound (or enzyme) capable of degrading dNTPs.

TABLE 4 Set-up of experiment and order of nucleotide addition. dATP dCTPdGTP dTTP T1 0 (3:C; 1:A)4 1 Run-off (25) T2 0 0 4 Run-off (26) T3 0 0 0Run-off (30) T4 4 0 2 Run-off (24)

DNA Sequencing Using Microfluidics on Sensor Chip. Sequencing in theflow regime is an extension of open application of nucleotide reagentsfor incorporation into DNA. Rather than add the reagents into a bulksolution on the ISFET chip, the reagents are flowed in a sequentialmanner across the chip surface, extending a single DNA base(s) at atime. The dNTPs are flowed sequentially, beginning with dTTP, then dATP,dCTP, and dGTP. Due to the laminar flow nature of the fluid movementover the chip, diffusion of the nucleotide into the microwells andfinally around the nucleic acid loaded bead is the main mechanism fordelivery. The flow regime also ensures that the vast majority ofnucleotide solution is washed away between applications. This involvesrinsing the chip with buffer solution and apyrase solution followingevery nucleotide flow. The nucleotides and wash solutions are stored inchemical bottles in the system, and are flowed over the chip using asystem of fluidic tubing and automated valves. The ISFET chip isactivated for sensing chemical products of the DNA extension duringnucleotide flow.

Example 2 Low Ionic Strength Effects on Polymerase Activity

Experiments were conducted to test various polymerases for certainfunctionalities associated with the sequencing aspects and embodimentsdiscussed herein, including in particular low ionic strength nucleotideincorporation reactions. These functionalities include the ability of apolymerase to extend a sequencing primer in low ionic strength, affinityof a polymerase for primer/template nucleic acid hybrids in low ionicstrength, rate of primer extension, and ability of polymerase to extendprimers in the context of a well situated above a chemFET sensor. Eachof these functionalities will be discussed below.

Polymerase Activity in Low Ionic Strength. Polymerase activity can bemeasured by determining the extent to which a primer is extended along atemplate. A fluorescent reporter assay was devised to measure polymeraseactivity (FIG. 72A). Briefly, a fluorescently-labeled DNAoligonucleotide (referred to in the context of this Example as thetemplate) is attached to superparamagnetic beads via astreptavidin-biotin interaction. The 5′ end of the template is labeledwith biotin and the 3′ end is labeled with for example 6-FAM. The beadsare labeled with streptavidin. A DNA oligonucleotide primercomplementary to the 3′ end of the template is annealed to the template,and the beads are incubated with polymerases in an extension solutioncontaining buffer, salt and dNTP for sufficient time for extension ofthe primer based on the template sequence. After the incubation thepolymerase and other reaction components are removed by serial washingof the beads. If the primer is extended through a predeterminedrestriction enzyme recognition site, the fluorescently-labeled doublestranded DNA will be released when incubated with the properendonuclease (e.g., HindIII). The supernatant from the restrictiondigestion is then subjected to fluorescence quantification. Themagnitude of the released fluorescence is proportional to the amount ofDNA released by restriction endonuclease activity, which is proportionalto the amount of double-stranded DNA present on the bead, which in turnis proportional to the polymerase activity.

In particular, the data shown in FIGS. 72B and 72C was generated bybinding polymerases to bead-immobilized, primer/template hybrids in theabsence of dNTPs. After washing away unbound polymerase and bindingsolution components, the indicated extension mix (e.g., standard or lowionic strength) supplemented with dNTPs was added to the beads andincubated for 10 minutes at 40° C. The activity assay was subsequentlycompleted as described above to measure the activity of the indicatedpolymerases and the background subtracted fluorescence is plotted. InFIG. 72C “Therminator(+)” indicates the addition of dNTPs in theextension solution, and “Therminator(−)” indicates the absence of dNTPsin the extension solution i.e. a positive and negative control,respectively.

Using this assay polymerase activity has been measured in both standardand low ionic strength reaction conditions. A number of polymerases arerelatively active in low ionic strength conditions when compared tostandard conditions (FIGS. 72B and 72C).

Polymerase Affinity in Low Ionic Strength. The affinity of a polymerasefor a template/primer hybrid can be measured indirectly with ourstandard assay (FIG. 72A). Three different DNA polymerases (T4exo−,Therminator, Klenow exo−) were bound to a primer-activated,bead-immobilized template (FIG. 72A, step 1) in a standard extensionsolution without dNTP for 15 minutes at room temperature. Beads are thenwashed to remove unbound polymerase and other components, and thenresuspended and incubated in a low ionic strength solution (e.g., 0.5 mMTris, 100 μM MgCl₂, pH 9.0 at 25° C.) at 40° C. After the timesindicated in FIG. 73, the beads were washed and resuspended in low ionicstrength solution (e.g., 0.5 mM Tris, 100 μM MgCl₂, pH 9.0 at 25° C. forTherminator and Klenow exo− or 0.5 mM Tris, 80 μM MgCl2, pH 9.0 at 25°C. for T4 exo−) supplemented with 20 μM each dNTP. If the polymerase hasa low affinity for the template/primer hybrid it would be expected tofall off and be washed away, and as a result there would be no primerextension. However, if the polymerase has a high affinity for thetemplate/primer hybrid it would remain bound and could extend thetemplate when incubated with dNTP. The amount of extended primer ismeasured using our standard assay (FIG. 72A) and is plotted in FIG. 73.Primer extension was equivalent at all time points suggesting that allthree polymerases bind tightly to the primer-activated template for 40minutes when incubated at 40° C. As a positive control (indicated as“(+)dNTP”) primer extension without prebinding of the polymerase toprimer-activated template was measured, and as a negative control(indicated as “(−) dNTP”) fluorescence was measured if dNTPs were notadded to extension solution. T4 exo− polymerase (3′ to 5′ exonucleasedeficient), Therminator, and Klenow exo− extend primer after incubationfor 40 minutes in low ionic strength solution suggesting that all threepolymerases have a high affinity for template/primer hybrid in low ionicstrength (FIG. 73).

Rates of Polymerases. The rate at which a polymerase can extend a primeralong a template can be measured with a standard assay by measuring thefraction of template extended per unit time. T4 exo− polymerase (3′ to5′ exonuclease deficient) extends primer in both standard reactionconditions (STD; 10 mM Tris-HCl, 50 mM NaCl, 10 mM MgCl₂, 1 mMdithiothreitol, 20 μM each dNTP, pH 7.9 at 25° C.) and in low ionicstrength conditions (LS; 0.5 mM Tris, 80 μM MgCl₂, 20 μM each dNTP, pH9.0 at 25° C.), but at different rates. Previous studies of T4demonstrate that T4 can extend a primer along a template at a maximalsteady state rate of 4 nucleotides per second in a standard reactionsolution (i.e., at standard ionic strength). At this rate, extension inthe standard reaction solution is expected to be complete in 8.75seconds, and indeed extension is complete at about 10 secondsexperimentally (FIG. 74A, T4-STD). In low ionic strength reactionsolution T4 completes extension in 20 seconds suggesting a two folddecrease. Furthermore, when the reaction solution is 0.5 mM Tris, 20 μMMgCl₂ and 5 μM of each dNTP the rate of T4 decreases by about twofoldrelative to 80 μM MgCl₂ and 20 μM for each dNTP (i.e., the low ionicstrength solution of FIG. 74A, see FIG. 74B) and fourfold relative to 10mM MgCl₂ and 20 μM for each dNTP (i.e., the standard solution of FIG.74A, data not shown). T4 has been reported to polymerize with biphasickinetics including an initial burst of polymerization (or incorporation)followed by a slower polymerization rate. This initial burst ofpolymerization can compensate for the slightly reduced rate observed atlow ionic strength. (Capson et al. Biochemistry 1992 31:10984-10994.)

Therminator and Bst polymerases can extend primer in both standardreaction conditions (STD; 20 mM Tris-HCl, 10 mM (NH2)₂SO₄, 10 mM KCl, 2mM MgSO₄, 0.1% Triton X-100, pH 8.8 at 25° C.) and in low ionic strengthconditions (LS; 0.5 mM Tris, 100 μM MgCl₂, 20 μM each dNTP, pH 9.0 at25° C.). The steady state rate in low ionic strength conditions isslower than extension by these polymerases in standard ionic strengthconditions, and it is slower than the steady state rate of T4 primerextension in low ionic strength extension solutions.

Primer Extension in Wells of Sensor. Using an assay similar to thestandard activity assay (FIG. 72A) primer extension was tested in thesilicon wells of a chemFET sensor to determine whether or not the sensorwell wall material inhibits the polymerase. This assay differs fromthose described above only in that the template is not labeled with6-FAM. Beads with polymerase-bound primer-activated template werepipetted onto the silicon dye (as shown in FIG. 75A), incubated for 1minute at 40° C. followed by the addition of dNTPs in a low salt buffer.After the incubation the beads are recovered from the chip and subjectedto a standard restriction digest. Primer extension was measured by DNAfragment separation using a BioAnalyzer. The addition of dNTPs resultedin a fragment whereas the reaction without dNTPs did not, suggestingthat the sensor well wall material does not inhibit polymerase extensionof the primer (FIG. 75B).

Example 3 Increasing Speed and Throughput

One advantage of the array architecture used on the ISFET/chemFET chipsis that all microwells receive and process chemical materials inparallel and the outputs of the FETs are analyzed in parallel. While asingle chip is limited to N wells and the number N will increase overtime as semiconductor processes shrink devices ever smaller, no matterhow many FETs and wells (also known as reaction chambers) are on asingle chip, a system may readily be build with two or more chips in thesame machine, operated in parallel; or multiple machines may be operatedin parallel and as their FETs provide output, those outputs can beprocessed in parallel to reduce the time required to sequence a genomeor perform another analysis. Parallel computing is well known and thereare multiple known ways for computer scientists to configure theprocessing of the outputs of several chips to expedite the computationof the desired output. Hence, with multiple chips, each having 256 k ormore microwells, it is readily achievable to extract from a sample ahuman genome in a span of one or just a few hours. As chip speedsincrease and device sizes shrink, these times may be achievable with asingle chip.

Having explained the theory and objective of the ion pulse detectionapproach, above, it may be helpful to some to provide basic examples ofconditions that appear useful, based on simulation, in somecircumstances, for achieving detection of DNA sequencing reactions.

The ions in solution. The ionic strength is a factor that is involved indetermining the ion pulse response. It is helpful to limit the ions inthe reaction buffer. Assuming 0.5 mM Tris and use of CF to balance thecharge, the following may be determined:

Species Concentration (mM) Tris 0.452 Tris-H⁺ 0.048 Cl⁻ 0.048

Consider the case of 0.5 mM Tris, as other concentrations of Tris (e.g.5-20 mM) are given by scaling the above concentrations. Assume the pH isconstant at 8.8. The other species in the solution include the reactantsand products:

Species Concentration dynamics (μM) dNTP⁻⁻⁻⁻ 0 → 6.5 H⁺ 10^(−2.8) →transient → 10^(−2.8) PPi⁻⁻⁻ 0 → transient → 0 Pi⁻⁻ 0 (if no PPi → Pienzyme present)This is the situation for the reaction buffer, scaled to 0.5 mMTris-HCl. It is assumed that all the Mg⁺⁺ is complexed to the enzyme bypresoaking. From the solution of the reaction and diffusion equation forthe sequencing reaction, the ionic strength is calculated, I=½Σ_(s)q_(s)²C_(s), and the sensor dynamics is given by relationships set out above.

Since the concentration changes are small, it is possible to carry outan expansion of the ionic strength in concentration changes:I={119.227+6.67[PPi]+2.78[Pi]+0.79[H+ released]−9.52(6.5μM−[dNTP])}×10⁻⁶  (5)The pH is 8.80.

For BST reaction buffer at 0.5 mM Tris, which contains ions and sulfuricacid:I={1452.46+6.5[PPi]+2.87[Pi]+0.98[H+ released]−9.98(6.5μM−[dNTP])}×10⁻⁶  (5′)

For a 5 mM Tris buffer, the ionic strength is given byI={555.803+7.43[PPi]+2.92[Pi]+0.98[H+ released]−9.90(6.5μM−[dNTP])}×10⁻⁶  (5″)The pH is 9.03.

For a 0.5 mM Tris plus 1 mM MgCl₂, the ionic strength is given byI={3119.86+6.94[PPi]+2.78[Pi]+0.91[H+ released]−9.49(6.5μM−[dNTP])}×10⁻⁶  (5′″)The pH is 8.81.

For a 5 mM Tris plus 1 mM MgCl₂, the ionic strength is given byI={3556.67+7.71[PPi]+2.93[Pi]+0.98[H+ released]−9.89(6.5μM−[dNTP])}×10⁻⁶  (5″″)The pH is 9.04.

For 0.5 mM Tris, the ionic strength is given byI={250.699+6.44[PPi]+2.83[Pi]+0.96[H+ released]−9.67(20μM−[dNTP])}×10⁻⁶  (5′″″)The pH is 8.53 with the dNTP present and 8.97 without.

For 0.5 mM Tris plus 50 μM MgCl₂, the ionic strength is given byI={400.759+6.46[PPi]+2.84[Pi]+0.96[H+ released]−9.66(20μM−[dNTP])}×10⁻⁶  (5″″″)The pH is 8.53 with the dNTP present and 8.97 without.

Finally, consider a canonical buffer with pH=9 and ionic strength=300 mMin the absence of dNTP. The equation would beI={493.20+6.46[PPi]+2.84[Pi]+0.96[H+ released]−9.66(20μM−[dNTP])}×10⁻⁶  (5′″″″)

Note that the signal is proportional to I^(−1/2), so it is desirable tokeep the ionic strength of the reaction buffer solution as low aspossible. Note that (I₀+dI)^(−1/2)=I₀ ^(−1/2)−½(dI/I₀)I₀ ^(−1/2). Thus,the relative change to the signal is −½(dI/I₀). By comparing Eqs.5-5′″″″, one sees that dI is roughly independent of the buffer ionicstrength. Thus, if the initial ionic strength, I₀, is lower, therelative signal change is larger.

Reaction/Diffusion Calculation

The reactions that occur ared[DNA]/dt=−k _(bst)[dNTP][DNA]d[PPi]/dt=k _(bst)[dNTP][DNA]d[dNTP]/dt=k _(bst)[dNTP][DNA]

If the enzyme for the reaction PPi+H₂0→2Pi is present, then thisreaction is assumed to occur instantaneously. All of these speciesdiffuse with individual diffusion coefficients, except the DNA. The H⁺released by adding one more base of DNA, however, does diffuse.

A finite element method may be used to calculate the reaction anddiffusion of these species. The method preferably is encoded incylindrical coordinates. Reaction in each element follows the abovescheme, with the note below about the effect of Mg⁺⁺ on the reactionrate. In addition, the diffusion of Mg⁺⁺ is also tracked. Recall that inthe current calculations, however, Mg⁺⁺ is assumed to be constant.

The numerical code was a check against the analytical solution fordiffusion into a cylinder of diameter D and depth H:C(r,z,t)=c0−c0(2/π)Σ_(n=0) ^(∞) [e ^(−D(n+1/2)^2π^2t/H^2/() n+½)] sin[(n+½)πz/H]

The results agreed to 0.1% accuracy if a grid of (D/2)/100 points in ther direction and H/100 points in the z direction are used.

To determine the parameter k_(bst), the results from the Rothberg et alNature paper were used. That paper presented a simplified, mass-transfercalculation of the relevant diffusion, from which the relevant reactionsared[DNA]/dt=−k _(bst)[dNTP][DNA]d[PPi]/dt=k _(bst)[dNTP][DNA]−k _(c)([PPi])d[dNTP]/dt=k _(bst)[dNTP][DNA]+k _(c)([dNTP]₀−[dNTP])Summing the last two reactions, one findsd([PPi]+[dNTP])/dt=−k _(bst)[dNTP][DNA]−k _(c)([dNTP]₀−[PPi]−[dNTP])

These equations may be solved numerically to determine the values ofk_(bst) and k_(c), yielding k_(bst)=2.38 (μM s)⁻¹ and k_(c)=0.2 s⁻¹ at30° C.

The PPi→Pi Enzyme

The reaction PPi+H₂0→2Pi occurs at a finite rate. The rate constant inthe presence of the enzyme and Mg⁺⁺ is about 10³ s⁻¹ (Biochemistry 41(2002) 12025). So, this reaction is over in 1 ms. Thus, one can say thisconversion is instantaneous. However, in the absence of any enzyme orionic catalyst, the rate constant is about 3⁻¹ years⁻¹ (J. Chem. Soc.Farady Trans, 93 (1007) 4295). In this case, the reaction does notproceed at all. Many ions, however, do catalyze this reaction to someextent. Thus, if this reaction is desired not to proceed, care must betaken that the solution is chosen so that no ions catalyzing thereaction are present.

Diffusion Coefficients

The signal due to the pH change from the sequencing reaction depends onthe diffusion coefficients of the species involved. Reference (Biophys.J 78 (2000) 1657) gives values for Pi, PPi, and dNTP:

Species 10⁶ D (cm²/s) at 37° C. Pi 9.5 PPi 7.48 ATP 5.3

Reference (BBA 1291 (1996) 115) gives values for ATP over a temperaturerange. At 37° C., the value is 4.5×10⁻⁶ cm²/s, which is used herein. Thevalues for Pi and PPi are calculated at temperature T by scaling thevalue above by the factor D_(ATP)(T)/D_(ATP)(37° C.). The diffusioncoefficients of the ions at 25° C. are (J. Chem. Phys. 119 (2003)11342.)

Species 10⁵ D (cm²/s) at 25° C. Mg++ 1.25 (J. Amer. Chem. Soc. 77 (1955)265) Na+ 1.33 (J. Chem. Phys. 119 (2003) 11342.)

The diffusion coefficient of H⁺ is assumed equal to that of Na⁺, becauseby charge neutrality the H⁺ must either diffusion with a negative ion asa pair, or the H⁺ must exchange with another positive ion, of which theN⁺ is the most rapidly diffusing.

One preferred implementation includes the use of small spheres (e.g. 0.1μm) to decrease the diffusion coefficients of all the species. Thisshifts the pH curves to larger time by roughly the ratio that thediffusion coefficient is reduced. The textbook formula for the reductionof the diffusion coefficient isD _(eff) /D ₀=2(1−φ)/(2+φ),  (6)where φ is the volume fraction occupied by the spheres. The radius ofthe small spheres does not directly enter into this expression. For nospheres φ=0. For randomly packed spheres, φ≈0.6, and for close packedspheres, φ=0.74.

Another approach is to use other methods to reduce the diffusioncoefficient of the H⁺, Pi, PPi, and dNTP. For example, the viscosity ofthe solution may be increased.

The Sequencing Reaction

Now consider the effect of the Mg⁺⁺ diffusing in. It is not knownexactly how the enzyme reaction rate depends on [Mg⁺⁺]. However, adivalent metal ion is required for enzymatic activity. It is assumedthat the rate is linearly proportional up to a maximum rate at 1 mM ofMg⁺⁺. Thus, it is assumed the reaction rate constant is equal tok _(bst)min(1,[Mg⁺⁺]/1 mM)  (7)

One may use a diffusion coefficient for Mg⁺⁺ of 1.25×10⁻⁵ cm²/2. ForModel 2, at t=0, the Mg⁺⁺ is only at z=0, at concentration [Mg⁺⁺]₀=1 mM.For t>0 it diffuses in and affects the reaction rate by Eq. (8). ForModel 1, the concentration of Mg⁺⁺ is 1 mM at all times in the well.Alternatively, we may assume that the enzyme is presoaked with Mg⁺⁺, sothat the concentration of free Mg⁺⁺ in solution is zero.

In the range of 7-9 pH, the reactions release H⁺ roughly as follows

DNA_(n) n DNA_(n+1) n + 1 dNTP 4 PPi 3 2 Pi 4This is because all the H⁺ with pKa values lower than the pH of thesolution will come off, to a good approximation. All of these specieslower the pH if the solution is pH 7 to 9. So the net effect of thereactiondNTP+DNA_(n)→PPi+DNA_(n+1)  (8)is to not change the pH.

The net effect of the reactionPPi+H₂O→2Pi  (9)is to change the pH by release of one net H⁺.

The H⁺ released by the DNA on the bead surface will diffuse out tobeyond the DNA, and since the pH is much higher than the pKa of the DNA,these H⁺ ions will diffuse out of the well during the equilibration timet<0. (All these H⁺ diffuse away.) That is, if the DNA on the bead isallowed to equilibrate before the dNTP is added, the solution shouldjust be the usual buffer solution (with a bit of extra Na⁺ near the beadto balance the negative charge on the DNA, within the Debye length).When the extra base of DNA is added, a pulse of H⁺ will come off—with adiffusion coefficient of something like Na⁺.

Well Sizes

Simulation runs were done for small wells:

D(μm) H(μm) 2 R_(bead) (μm) V_(well) (pl) [DNA]_(well) (μM) 1.5 2.251.05 0.005063 4.62 4 6 2.8 0.096 1.73 6 8 5.91 0.29 2.55

For D=1.5 μm, h=0.2 μm (1 layer of beads). For D=4 μm, h=1.1 μm.Otherwise h=1.5 μm. In addition, one run was done for the 4 μm×6 μmwells with a larger bead of radius 3.6 μm.

dNTP Front Thickness in Device Flow

The dNTP is assumed in the calculations to instantaneously change from 0to 6.5 μM above the wells at t=0. This condition is approximately trueif the dNTP flows quickly enough past the wells. Define

-   -   V=velocity of flow of the fluid above the well    -   L=length of device    -   D=diffusivity of dNTP=3.91×10⁻⁶ cm²/s    -   d=size of well    -   t=time to sweep flow across device    -   t₀=cutoff time for “instantaneous change”    -   W_(d)=width of front due to diffusion at end of        device=(2Dt)^(1/2)    -   W_(m)=width of front due to mixing≈length of flush volume region    -   U=flush volume=15 μl (currently 52 μl)    -   u=flow rate=50 μl/s

Flow cell geometries are as follows for two models, designated 313 and314:

Flow axis Width Height Volume Cell Cell (mm) (mm) (mm) (μl) Volumes/s314 7.249 7.249 0.336 17.657 2.775 313 4.999 4.999 0.293 7.321 6.693

Take L=5 mm, d=4 μm and assume t=1/6.69 s. Q=50 μl/s. Then W_(d)=10.8μm. Also, V=L/t=3.3 cm/s. D_(eff)=D₀2(1−φ)/(2+φ)=1.20×10⁻⁶ cm²/s forφ=0.6. The value of t₀ depends on the well size. It should be muchshorter than the time at which dNTP diffuses to the bottom of the wellfor N_(poly)=0 (i.e. much shorter than the time at which this ion pulseresponse occurs), say 10% of that value. For the 4×6 μm well the timefor the dNTP to diffuse to the bottom of the well, assuming noobstruction to the diffusion is H²/(2D), so that t₀=10%×H²/(2D)=0.015 s.Simulation reveals the sphere with the DNA on it slows down thediffusion, so that perhaps t₀=10%*0.2 s=0.02 s. Define W=max(W_(d),W_(m)). The time the leading edge of the front takes to cross the deviceis d/V. The time it takes the whole front (leading plus trailing edge)to cross a well is (W+d)/V. The condition of “instantaneous” rise of thedNTP concentration is (W+d)/V<<t₀. (W+d)/V≈U/u. Since U/u=0.3 s, andt₀=0.02 s, this condition is not satisfied by the above assumptions.Either U must be made smaller or u must be made larger, or both, by afactor of 10. The condition that W_(m) is given by the length of theflush region may be a bit too strict: the width of the mixing region maybe substantially smaller than the length of the entire flush region.This is a conservative condition. However, W_(d)/V<<t₀, so diffusivespreading of the front can be ignored.

Now, a different calculation. It is desirable for the concentration ofdNTP at the wall of the channel to rise from 0 to 6.5 μM in less thant₀. The concentration C(x,y,z) at t=0 may be assumed to be c₀=6.5 μM att=0. Take 0<x<L, −B<y<B, 0<z<W. It is desired to know the concentrationat y=B for any value of 0<x<L. Strictly speaking, the concentration atthe wall stays 0, since the velocity there is zero, unless diffusion isconsidered. So, it is necessary to consider convection in the xdirection and diffusion in the z direction. Calculating the convection[6]v=[(p ₀ −p _(L))B ²/(2 μL)][1−(y/B)²]  (10)

The flow rate is Q=(⅔)(p₀−p_(L))B³W/(μL). Calculate the time it takesfor the average flow at the end of the device to be 0.95c₀. First, notethat without diffusion, the concentration is given byc(x,y,z)=0, t<t*c ₀ , t>t*t*=x/v(y)  (11)The flow becomes 95% of the maximum concentration at t′=L/v(0.95B). Thetime to clear one volume is t″=volume/Q, where volume=2BWL. It can befound that t′=⅔/0.0975t″=6.87t″. Thus it takes 7 device volumes to bringthe average concentration up to 95% of the final value (not 3). Ofconcern is the value of the concentration at the device wall. Theconcentration obeys the equation∂c/∂t+v(y)∂c/∂x=D∂ ² c/∂y ²  (12)c(x,y,0)=0c(0,y,t)=c ₀∂c(x,±B,t)/∂y=0ignoring diffusion in the y direction as dominated by convection. Nowthe concentration reaches the wall by diffusion in a time δt, butdiffusion from the concentration profile that existed at a smaller x−δxand y=y−δy, since convection occurs during this time δt. Note (δy)²=2Dt.From the above, it is known that the concentration can diffuse on theorder of δy=10 μm by diffusion during a time t″=1/6.69 s. It is desiredthat it diffuse during a time t₀=0.02 s, which implies a diffusiondistance of 3.95 μM. So, as a conservative calculation, one couldcalculate from Eq. (11) t*=L/v(B−3.95 μm). By the time t*+t₀, theconcentration at the wall will be approximately c₀ through a combinationof diffusion and convection. Thusv=(¾)(1/BW)Q[1−(y/B)^2]Using this, t*=L/v(B−3.95 μm)=1.84 s. At a value t₀ later, theconcentration at the wall will rise approximately to the maximum.Unfortunately, this calculation ignores that during 1.84 s, materialfrom velocity profiles from smaller values of y will also have had thechance to diffuse to the wall. It is also ignoring that the diffusionwhich starts for smaller values of x proceeds to the right with lowervelocities as y gets larger.

Thus, one can ask when diffusion over the boundary layer is faster thanconvection. By diffusion δ/t=2D/δ. By convection δ/t=v(B−δ). These twoare equal for δ=0.001 mm=1.0 μm. So, when the front has reached thisvalue of δ=L/v(B−δ)=6.78 s, the progression of the material to the wallis entirely dominated by diffusion. And the concentration will rise toapproximately c₀ during the time δ²/(2D)=0.01 s. So, for δ<1.0 μm,convection is more important than diffusion in the x direction.

A numerical solution of Eq. (12) is necessary to check the aboveapproximate calculations. A finite element method can be used tonumerically solve Eq. (12). We find that for the above device, theconcentration at the wall rises in about 0.15 s at x=0.5 mm and about0.5 s at x=5.0 mm. This is too slow. The Reynolds number is Re=1g/cm³*0.5 cm/(1/6.69 s)*0.0293 cm/0.01 cp=9.8. If one sets the height tobe 0.1 mm, one finds that the concentration at the wall rises in about0.04 s at x=0.5 mm and about 0.15 s at x=5.0 mm. This is probably tooslow. Re=9.8. If set alternatively set Q=240 μl/s, the concentration atthe wall rises in about 0.05 s at x=0.5 mm and about 0.2 s at x=5.0 mm.This is probably too slow. Re=47. If one sets both the height to be 0.1mm and Q=240 μl/s, the concentration at the wall rises in about 0.013 sat x=0.5 mm and about 0.07 s at x=5.0 mm. This is probably fast enough.Re=47. The flow in all cases should be laminar.

The above may be a bit too strict, in that the biggest signal is comingfrom the breakthrough of the dNTP, and this breakthrough is quite sharp.It is possible that the dNTP rise at the top of the well need not becomplete in 0.02 s. Perhaps the rise just has to be completesignificantly before the dNTP breakthrough at the bottom of the welloccurs. And this is on the order of seconds. So, one needs to solve thereaction/diffusion equations with a dNTP concentration that is rising atthe top of the well. Let it rise from 0 to 6.5 μM linearly between 0 andt, where t is the value specified in the previous paragraph for the risetime at the end of the device.

From these numerical simulations, it may be found that a finite risetime of the dNTP at the surface of the well reduces the performance ofthe ion pulse device only slightly. For a 6 μm×8 μm device, a finiterise time of 0.2 s affects the observed sensor response curves onlyslightly. A finite rise time of 0.5 s affects the observed sensorresponse curves by shifting to longer times the first peak as well asreducing the height of the peaks for all values of N_(poly). A surfacewith a slower response time would be less affected by this finite dNTPrise time. The effects are similar, if a bit more dramatic, for the 4μm×6 μm, since the characteristic diffusion times for this device aresmaller and therefore the finite rise time is a larger relativeperturbation to the idealized case of instantaneous dNTP rise at thesurface of the well.

Parametric Sensitivity of Results

To determine the sensitivity of the ion pulse response to experimentalvariables, the 4 μm×6 μm system was taken as a reference. Since thisdetection method relies on diffusion, the well geometry has asignificant impact on the resolution of any N_(poly) curves that areplotted.

The dNTP concentration was halved to 3.25 μM and doubled to 13 μM. TheDNA on the bead surface was increased by a factor of 3. A run was donewith a larger 3.6 μm bead. Both the DNA on the bead surface and the dNTPwere increased by a factor of 3. The reaction rate, k, of the enzyme washalved and doubled. Sensor response curves for the 5 μM Tris, no freeMg⁺⁺, were produced for all of the above.

Double the enzyme reaction rate constant makes the peaks very slightlyhigher.

One possibility is that the DNA concentration on the beads is so highthat the reaction is effectively diffusion limited, and so the enzymerate constant is not a major factor affecting the concentration curves.Similarly, reducing the enzyme reaction rate by ½ has little effect onthe sensor curves. It spreads them out in time only very slightly.

The large bead works very well. Since there is more mass of DNA on thebead, consuming it takes longer. In addition, the bead is taking up morespace in the volume, so that diffusion to the well bottom takes a bitlonger. The peaks are spread out to larger times.

A larger concentration of DNA on the bead spreads out the peaks in time.This is because the larger concentration of DNA means a greater mass ofDNA on the beads. This greater mass of DNA takes a longer time toreaction. Thus the final breakthrough of the dNTP takes longer.

Lowering the solution dNTP concentration to 3.25 μM spreads out thepeaks in time, but also reduces the peak height. The two effectsprobably approximately cancel in terms of distinguishing the differentN_(poly) curves. Conversely, increasing the dNTP concentration to 13 μMshifts the peaks to smaller time. While the peaks are higher, they arealso substantially closer in time. The curves are at such a shorter timethat they are running into the response time of the sensor. It appearsthe 13 μM curves may be less distinguishable than the 6.5 μM case.

Increasing the concentration of DNA on the beads three times andincreasing the dNTP concentration in the solution to 13 μM makes thepeaks about 3 times higher but does not spread them out in time. Thetime scale is relatively unchanged because while the amount of DNA onthe beads is greater, the reaction rate is also greater due to thehigher dNTP concentration. The peaks are higher because there is moremass released in the same time.

Packing Beads—Φ (Phi)

The desirability for packing beads (to slow down diffusion of allspecies) is seen by comparing the Ion Pulse Response between similarwell dimensions and between the 4×6 micron (width×height) and 6×8 micronwells. The N_(poly) curves resolve with increased depth of the well andwith packing beads (Φ=0.6).

The formula to see the effect of Φ is the effective diffusioncoefficientD _(eff) =D ₀*2(1−Φ)/(2+Φ)

For Φ=0.6, D₀/D_(eff)=3.25 e.g. 3.25× slowdown due to beads

For Φ=0.9, D₀/D_(eff)=14.5 e.g. 14.5× slowdown due to this

The other formula needed is the characteristic time in the wellH²=2D_(eff) tSo, for a 6 um well at Φ=0.6t=6²/(2/3.25*D ₀)=58.5/D ₀A 2.25 well at Φ=0.9 yieldst=2.25²/(2/14.5*D ₀)=36.7/D ₀So, the 2.25 um well at Φ=0.9 would have all times about 36.7/58.5=0.63smaller than the 6 um well at Φ=0.6. The smaller well will still be alittle bit faster. If one had Φ=0.936, the smaller well would operatejust about exactly as the larger well (assuming the well diameter andthe DNA bead are scaled the same way as the well height).

Signal to Noise

The signal-to-noise ratio is proportional to the difference in mVbetween a N_(poly) and N_(poly+1) signal and also to the square root ofthe length of time over which the mV curves are different. Define σ asthe sensor noise and f as the number of frames per second. Define theaverage difference of the absolute value between signal 1 and signal 2during 0<t<t_(max) as Δ, i.e.Δ=∫₀ ^(tmax) |v ₁ −v ₂ /t _(max).

Take the absolute value because the difference between the curves canhave both signs. Thus the condition isΔ>σ[2/(ft _(max))]^(1/2)

Consider, as an example, for the D=4 μm, H=6 μm wells, φ=0, dNTP=20 μM,10×DNA case. Take f=20 Hz, and t_(max)=2 s. The value of Δ is 0.6861.Thus0.6861>0.22σ

So, using the value of σ=0.25 mV, this device will work (1−P=10⁻³⁵).Consider as another example the D=4 μm, H=6 μm wells, φ=0, dNTP=6.5 μM,10×DNA case. Take f=20 Hz, and t_(max)=4 s. The value of Δ is 0.1862.Thus0.1862>0.1581σ

So, using the value of σ=0.25 mV, this device will probably work(1−P=2.4×10⁻⁶).

Note there is an optimal value of t_(max): too small a value, and onedoes not have enough signal; too large a value, and one is seeing mostlynoise. The value of t_(max) is only roughly here.

EQUIVALENTS

While several inventive embodiments have been described and illustratedherein, those of ordinary skill in the art will readily envision avariety of other means and/or structures for performing the functionand/or obtaining the results and/or one or more of the advantagesdescribed herein, and each of such variations and/or modifications isdeemed to be within the scope of the inventive embodiments describedherein. More generally, those skilled in the art will readily appreciatethat all parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the inventive teachingsis/are used. Those skilled in the art will recognize, or be able toascertain using no more than routine experimentation, many equivalentsto the specific inventive embodiments described herein. It is,therefore, to be understood that the foregoing embodiments are presentedby way of example only and that, within the scope of the appended claimsand equivalents thereto, inventive embodiments may be practicedotherwise than as specifically described and claimed. Inventiveembodiments of the present disclosure are directed to each individualfeature, system, article, material, kit, and/or method described herein.In addition, any combination of two or more such features, systems,articles, materials, kits, and/or methods, if such features, systems,articles, materials, kits, and/or methods are not mutually inconsistent,is included within the inventive scope of the present disclosure.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

All references, patents and patent applications disclosed herein areincorporated by reference with respect to the subject matter for whicheach is cited, which in some cases may encompass the entirety of thedocument.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Multiple elements listed with“and/or” should be construed in the same fashion, i.e., “one or more” ofthe elements so conjoined. Other elements may optionally be presentother than the elements specifically identified by the “and/or” clause,whether related or unrelated to those elements specifically identified.Thus, as a non-limiting example, a reference to “A and/or B”, when usedin conjunction with open-ended language such as “comprising” can refer,in one embodiment, to A only (optionally including elements other thanB); in another embodiment, to B only (optionally including elementsother than A); in yet another embodiment, to both A and B (optionallyincluding other elements); etc.

As used herein in the specification and in the claims, “or” should beunderstood to have the same meaning as “and/or” as defined above. Forexample, when separating items in a list, “or” or “and/or” shall beinterpreted as being inclusive, i.e., the inclusion of at least one, butalso including more than one, of a number or list of elements, and,optionally, additional unlisted items. Only terms clearly indicated tothe contrary, such as “only one of” or “exactly one of,” or, when usedin the claims, “consisting of,” will refer to the inclusion of exactlyone element of a number or list of elements. In general, the term “or”as used herein shall only be interpreted as indicating exclusivealternatives (i.e. “one or the other but not both”) when preceded byterms of exclusivity, such as “either,” “one of,” “only one of,” or“exactly one of.” “Consisting essentially of,” when used in the claims,shall have its ordinary meaning as used in the field of patent law.

As used herein in the specification and in the claims, the phrase “atleast one,” in reference to a list of one or more elements, should beunderstood to mean at least one element selected from any one or more ofthe elements in the list of elements, but not necessarily including atleast one of each and every element specifically listed within the listof elements and not excluding any combinations of elements in the listof elements. This definition also allows that elements may optionally bepresent other than the elements specifically identified within the listof elements to which the phrase “at least one” refers, whether relatedor unrelated to those elements specifically identified. Thus, as anon-limiting example, “at least one of A and B” (or, equivalently, “atleast one of A or B,” or, equivalently “at least one of A and/or B”) canrefer, in one embodiment, to at least one, optionally including morethan one, A, with no B present (and optionally including elements otherthan B); in another embodiment, to at least one, optionally includingmore than one, B, with no A present (and optionally including elementsother than A); in yet another embodiment, to at least one, optionallyincluding more than one, A, and at least one, optionally including morethan one, B (and optionally including other elements); etc.

It should also be understood that, unless clearly indicated to thecontrary, in any methods claimed herein that include more than one stepor act, the order of the steps or acts of the method is not necessarilylimited to the order in which the steps or acts of the method arerecited.

In the claims, as well as in the specification above, all transitionalphrases such as “comprising,” “including,” “carrying,” “having,”“containing,” “involving,” “holding,” “composed of,” and the like are tobe understood to be open-ended, i.e., to mean including but not limitedto. Only the transitional phrases “consisting of” and “consistingessentially of” shall be closed or semi-closed transitional phrases,respectively, as set forth in the United States Patent Office Manual ofPatent Examining Procedures, Section 2111.03.

What is claimed is:
 1. A method for nucleic acid sequencing, the methodcomprising: disposing at least one template nucleic acid in a reactionchamber communicating with a chemical sensor; introducing knownnucleotides into the reaction chamber; detecting an output pulse fromthe chemical sensor indicating an end of an incorporation interval ofthe known nucleotides into the template nucleic acid; and determining anumber of known nucleotides incorporated during the incorporationinterval based on a time interval between the introduction of the knownnucleotides and the detection of the output pulse.
 2. The method ofclaim 1, wherein the end of the incorporation interval defines an end ofincorporation events of the known nucleotides into the at least onetemplate nucleic acid.
 3. The method of claim 1, wherein the outputpulse is due to a change in ion concentration within the reactionchamber at the end of the incorporation interval.
 4. The method of claim3, wherein the change in ion concentration is due to a change inconcentration of unincorporated known nucleotides within the reactionchamber.
 5. The method of claim 4, wherein the change in ionconcentration is further due to a diffusion of ions out of the reactionchamber.
 6. The method of claim 1, wherein the incorporated knownnucleotides are of a single type.
 7. The method of claim 1, wherein thechemical sensor is coupled to the reaction chamber via a passivationlayer.
 8. The method of claim 7, wherein a rate of ion generation withinthe reaction chamber due to incorporation of the known nucleotides isgreater than a rate of change in surface charge density of thepassivation layer.
 9. The method of claim 1, wherein the chemical sensoris a chemically-sensitive field effect transistor (chemFET), and theoutput pulse is based on a threshold voltage of the chemFET.
 10. Themethod of claim 9, wherein the chemFET includes a floating gate coupledto the reaction chamber via a passivation layer.
 11. A method fornucleic acid sequencing, the method comprising: disposing at least onetemplate nucleic acid in a reaction chamber communicating with achemical sensor; introducing known nucleotides into the reactionchamber; detecting a first output pulse from the chemical sensorindicating a beginning of incorporation events of the known nucleotidesinto the template nucleic acid; detecting a second output pulse from thechemical sensor indicating an end of incorporation events of the knownnucleotides into the template nucleic acid; and determining a number ofknown nucleotides incorporated into the at least one template nucleicacid based on a time interval between the detection of the first outputpulse and the detection of the second output pulse.
 12. The method ofclaim 11, wherein the first pulse is due at least in part to generationof PPi within the reaction chamber resulting from the incorporationevents.
 13. The method of claim 12, wherein the second pulse is due atleast in part to a change in concentration of unincorporated knownnucleotides within the reaction chamber.
 14. The method of claim 11,wherein the first pulse and the second pulse are each due to changes inion concentration within the reaction chamber.
 15. The method of claim11, wherein the chemical sensor is coupled to the reaction chamber via apassivation layer.
 16. The method of claim 15, wherein a rate of iongeneration within the reaction chamber due to incorporation of the knownnucleotides is greater than a rate of change in surface charge densityof the passivation layer.
 17. The method of claim 11, wherein thechemical sensor is a chemically-sensitive field effect transistor(chemFET), and the output pulse is based on a threshold voltage of thechemFET.
 18. The method of claim 17, wherein the chemFET includes afloating gate coupled to the reaction chamber via a passivation layer.19. A method for nucleic acid sequencing, the method comprising:disposing at least one template nucleic acid in a reaction chambercommunicating with a chemical sensor; introducing known nucleotides intothe reaction chamber; and detecting incorporation of at least one of theknown nucleotides into the at least one template nucleic acid byreceiving a pair of output pulses from the chemical sensor in responseto introducing the known nucleotides.
 20. The method of claim 19,further comprising: introducing second known nucleotides into thereaction chamber; and detecting a lack of incorporation of the secondknown nucleotides by receiving a single output pulse from the chemicalsensor in response to introducing the second known nucleotides.